Table of Content
I.Project Summary
II.Workflow Checklist
III.NGS Sequencing Results
IV.Complete Report Download
V.Raw Sequence Data Download
VI.Analysis - DADA2 Read Processing
 Sample Meta Info
 Read Count by Sample
VII.Analysis - Read Taxonomy Assignment
 Taxonomy Barplots
VIII.Analysis - Alpha Diversity
IX.Analysis - Beta Diversity
X.Analysis - Differential Abundance
 ANCOM Result
 LEfSe Result
XI.Analysis - Heatmap Profile
XII.Analysis - Network Association

(Click to navigate)

FOMC Service Report

16S rRNA Gene V1V3 Amplicon Sequencing

Version V1.41

Version History

The Forsyth Institute, Cambridge, MA, USA
September 23, 2022

Project ID: FOMC7300

I. Project Summary

Project FOMC7300 services include NGS sequencing of the V1V3 region of the 16S rRNA gene amplicons from the samples. First and foremost, please download this report, as well as the sequence raw data from the download links provided below. These links will expire after 60 days. We cannot guarantee the availability of your data after 60 days.

Full Bioinformatics analysis service was requested. We provide many analyses, starting from the raw sequence quality and noise filtering, pair reads merging, as well as chimera filtering for the sequences, using the DADA2 denosing algorithm and pipeline.

We also provide many downstream analyses such as taxonomy assignment, alpha and beta diversity analyses, and differential abundance analysis.

For taxonomy assignment, most informative would be the taxonomy barplots. We provide an interactive barplots to show the relative abundance of microbes at different taxonomy levels (from Phylum to species) that you can choose.

If you specify which groups of samples you want to compare for differential abundance, we provide both ANCOM and LEfSe differential abundance analysis.

 

II. Workflow Checklist

☑1.Sample Received
☑2.Sample Quality Evaluated
☑3.Sample Prepared for Sequencing
☑4.Next-Gen Sequencing
☑5.Sequence Quality Check
☑6.Absolute Abundance
☑7.Report and Raw Sequence Data Available for Download
☑8.Bioinformatics Analysis - Reads Processing (DADA2 Quality Trimming, Denoising, Paired Reads Merging)
☑9.Bioinformatics Analysis - Reads Taxonomy Assignment
☑10.Bioinformatics Analysis - Alpha Diversity Analysis
☑11.Bioinformatics Analysis - Beta Diversity Analysis
☑12.Bioinformatics Analysis - Differential Abundance Analysis
☑13.Bioinformatics Analysis - Heatmap Profile
☑14.Bioinformatics Analysis - Network Association
 

III. NGS Sequencing

The samples were processed and analyzed with the ZymoBIOMICS® Service: Targeted Metagenomic Sequencing (Zymo Research, Irvine, CA).

DNA Extraction: If DNA extraction was performed, one of three different DNA extraction kits was used depending on the sample type and sample volume and were used according to the manufacturer’s instructions, unless otherwise stated. The kit used in this project is marked below:

☐ZymoBIOMICS® DNA Miniprep Kit (Zymo Research, Irvine, CA)
☐ZymoBIOMICS® DNA Microprep Kit (Zymo Research, Irvine, CA)
☐ZymoBIOMICS®-96 MagBead DNA Kit (Zymo Research, Irvine, CA)
☑N/A (DNA Extraction Not Performed)
Elution Volume: 50µL
Additional Notes: NA

Targeted Library Preparation: The DNA samples were prepared for targeted sequencing with the Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA). These primers were custom designed by Zymo Research to provide the best coverage of the 16S gene while maintaining high sensitivity. The primer sets used in this project are marked below:

☐Quick-16S™ Primer Set V1-V2 (Zymo Research, Irvine, CA)
☑Quick-16S™ Primer Set V1-V3 (Zymo Research, Irvine, CA)
☐Quick-16S™ Primer Set V3-V4 (Zymo Research, Irvine, CA)
☐Quick-16S™ Primer Set V4 (Zymo Research, Irvine, CA)
☐Quick-16S™ Primer Set V6-V8 (Zymo Research, Irvine, CA)
☐Other: NA
Additional Notes: NA

The sequencing library was prepared using an innovative library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore limit PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned up with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research, Irvine, CA), then quantified with TapeStation® (Agilent Technologies, Santa Clara, CA) and Qubit® (Thermo Fisher Scientific, Waltham, WA).

Control Samples: The ZymoBIOMICS® Microbial Community Standard (Zymo Research, Irvine, CA) was used as a positive control for each DNA extraction, if performed. The ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each targeted library preparation. Negative controls (i.e. blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process.

Sequencing: The final library was sequenced on Illumina® MiSeq™ with a V3 reagent kit (600 cycles). The sequencing was performed with 10% PhiX spike-in.

 

IV. Complete Report Download

The complete report of your project, including all links in this report, can be downloaded by clicking the link provided below. The downloaded file is a compressed ZIP file and once unzipped, open the file “REPORT.html” (may only shown as "REPORT" in your computer) by double clicking it. Your default web browser will open it and you will see the exact content of this report.

Please download and save the file to your computer storage device. The download link will expire after 60 days upon your receiving of this report.

Complete report download link:

To view the report, please follow the following steps:
1.Download the .zip file from the report link above.
2.Extract all the contents of the downloaded .zip file to your desktop.
3.Open the extracted folder and find the "REPORT.html" (may shown as only "REPORT").
4.Open (double-clicking) the REPORT.html file. Your default browser will open the top age of the complete report. Within the report, there are links to view all the analyses performed for the project.

 

V. Raw Sequence Data Download

The raw NGS sequence data is available for download with the link provided below. The data is a compressed ZIP file and can be unzipped to individual sequence files. Since this is a pair-end sequencing, each of your samples is represented by two sequence files, one for READ 1, with the file extension “*_R1.fastq.gz”, another READ 2, with the file extension “*_R1.fastq.gz”. The files are in FASTQ format and are compressed. FASTQ format is a text-based data format for storing both a biological sequence and its corresponding quality scores. Most sequence analysis software will be able to open them. The Sample IDs associated with the R1 and R2 fastq files are listed in the table below:



Sample IDOriginal Sample IDRead 1 File NameRead 2 File Name


F7300.S100Subject5.Visit3.Pzr7300_100V1V3_R1.fastq.gzzr7300_100V1V3_R2.fastq.gz


F7300.S101Subject6.Visit3.Pzr7300_101V1V3_R1.fastq.gzzr7300_101V1V3_R2.fastq.gz


F7300.S102Subject7.Visit3.Pzr7300_102V1V3_R1.fastq.gzzr7300_102V1V3_R2.fastq.gz


F7300.S103Subject8.Visit3.Pzr7300_103V1V3_R1.fastq.gzzr7300_103V1V3_R2.fastq.gz


F7300.S104Subject9.Visit3.Pzr7300_104V1V3_R1.fastq.gzzr7300_104V1V3_R2.fastq.gz


F7300.S105Subject10.Visit3.Pzr7300_105V1V3_R1.fastq.gzzr7300_105V1V3_R2.fastq.gz


F7300.S106Subject11.Visit3.Pzr7300_106V1V3_R1.fastq.gzzr7300_106V1V3_R2.fastq.gz


F7300.S107Subject12.Visit3.Pzr7300_107V1V3_R1.fastq.gzzr7300_107V1V3_R2.fastq.gz


F7300.S108Subject13.Visit3.Pzr7300_108V1V3_R1.fastq.gzzr7300_108V1V3_R2.fastq.gz


F7300.S109Subject15.Visit3.Pzr7300_109V1V3_R1.fastq.gzzr7300_109V1V3_R2.fastq.gz


F7300.S010zr7300_10V1V3_R1.fastq.gzzr7300_10V1V3_R2.fastq.gz


F7300.S110Subject16.Visit3.Pzr7300_110V1V3_R1.fastq.gzzr7300_110V1V3_R2.fastq.gz


F7300.S111Subject17.Visit3.Pzr7300_111V1V3_R1.fastq.gzzr7300_111V1V3_R2.fastq.gz


F7300.S112Subject18.Visit3.Pzr7300_112V1V3_R1.fastq.gzzr7300_112V1V3_R2.fastq.gz


F7300.S113Subject19.Visit3.Pzr7300_113V1V3_R1.fastq.gzzr7300_113V1V3_R2.fastq.gz


F7300.S114zr7300_114V1V3_R1.fastq.gzzr7300_114V1V3_R2.fastq.gz


F7300.S011Subject11.Visit1.Szr7300_11V1V3_R1.fastq.gzzr7300_11V1V3_R2.fastq.gz


F7300.S012Subject12.Visit1.Szr7300_12V1V3_R1.fastq.gzzr7300_12V1V3_R2.fastq.gz


F7300.S013Subject13.Visit1.Szr7300_13V1V3_R1.fastq.gzzr7300_13V1V3_R2.fastq.gz


F7300.S014Subject15.Visit1.Szr7300_14V1V3_R1.fastq.gzzr7300_14V1V3_R2.fastq.gz


F7300.S015Subject16.Visit1.Szr7300_15V1V3_R1.fastq.gzzr7300_15V1V3_R2.fastq.gz


F7300.S016Subject17.Visit1.Szr7300_16V1V3_R1.fastq.gzzr7300_16V1V3_R2.fastq.gz


F7300.S017Subject18.Visit1.Szr7300_17V1V3_R1.fastq.gzzr7300_17V1V3_R2.fastq.gz


F7300.S018Subject19.Visit1.Szr7300_18V1V3_R1.fastq.gzzr7300_18V1V3_R2.fastq.gz


F7300.S019Subject20.Visit1.Szr7300_19V1V3_R1.fastq.gzzr7300_19V1V3_R2.fastq.gz


F7300.S001Subject1.Visit1.Szr7300_1V1V3_R1.fastq.gzzr7300_1V1V3_R2.fastq.gz


F7300.S020Subject1.Visit2.Szr7300_20V1V3_R1.fastq.gzzr7300_20V1V3_R2.fastq.gz


F7300.S021Subject2.Visit2.Szr7300_21V1V3_R1.fastq.gzzr7300_21V1V3_R2.fastq.gz


F7300.S022Subject3.Visit2.Szr7300_22V1V3_R1.fastq.gzzr7300_22V1V3_R2.fastq.gz


F7300.S023Subject4.Visit2.Szr7300_23V1V3_R1.fastq.gzzr7300_23V1V3_R2.fastq.gz


F7300.S024Subject5.Visit2.Szr7300_24V1V3_R1.fastq.gzzr7300_24V1V3_R2.fastq.gz


F7300.S025Subject6.Visit2.Szr7300_25V1V3_R1.fastq.gzzr7300_25V1V3_R2.fastq.gz


F7300.S026Subject7.Visit2.Szr7300_26V1V3_R1.fastq.gzzr7300_26V1V3_R2.fastq.gz


F7300.S027Subject8.Visit2.Szr7300_27V1V3_R1.fastq.gzzr7300_27V1V3_R2.fastq.gz


F7300.S028Subject9.Visit2.Szr7300_28V1V3_R1.fastq.gzzr7300_28V1V3_R2.fastq.gz


F7300.S029Subject10.Visit2.Szr7300_29V1V3_R1.fastq.gzzr7300_29V1V3_R2.fastq.gz


F7300.S002Subject2.Visit1.Szr7300_2V1V3_R1.fastq.gzzr7300_2V1V3_R2.fastq.gz


F7300.S030Subject11.Visit2.Szr7300_30V1V3_R1.fastq.gzzr7300_30V1V3_R2.fastq.gz


F7300.S031Subject12.Visit2.Szr7300_31V1V3_R1.fastq.gzzr7300_31V1V3_R2.fastq.gz


F7300.S032Subject13.Visit2.Szr7300_32V1V3_R1.fastq.gzzr7300_32V1V3_R2.fastq.gz


F7300.S033Subject15.Visit2.Szr7300_33V1V3_R1.fastq.gzzr7300_33V1V3_R2.fastq.gz


F7300.S034Subject16.Visit2.Szr7300_34V1V3_R1.fastq.gzzr7300_34V1V3_R2.fastq.gz


F7300.S035Subject17.Visit2.Szr7300_35V1V3_R1.fastq.gzzr7300_35V1V3_R2.fastq.gz


F7300.S036Subject18.Visit2.Szr7300_36V1V3_R1.fastq.gzzr7300_36V1V3_R2.fastq.gz


F7300.S037Subject19.Visit2.Szr7300_37V1V3_R1.fastq.gzzr7300_37V1V3_R2.fastq.gz


F7300.S038Subject20.Visit2.Szr7300_38V1V3_R1.fastq.gzzr7300_38V1V3_R2.fastq.gz


F7300.S039Subject1.Visit3.Szr7300_39V1V3_R1.fastq.gzzr7300_39V1V3_R2.fastq.gz


F7300.S003Subject3.Visit1.Szr7300_3V1V3_R1.fastq.gzzr7300_3V1V3_R2.fastq.gz


F7300.S040Subject2.Visit3.Szr7300_40V1V3_R1.fastq.gzzr7300_40V1V3_R2.fastq.gz


F7300.S041Subject3.Visit3.Szr7300_41V1V3_R1.fastq.gzzr7300_41V1V3_R2.fastq.gz


F7300.S042Subject4.Visit3.Szr7300_42V1V3_R1.fastq.gzzr7300_42V1V3_R2.fastq.gz


F7300.S043Subject5.Visit3.Szr7300_43V1V3_R1.fastq.gzzr7300_43V1V3_R2.fastq.gz


F7300.S044Subject6.Visit3.Szr7300_44V1V3_R1.fastq.gzzr7300_44V1V3_R2.fastq.gz


F7300.S045Subject7.Visit3.Szr7300_45V1V3_R1.fastq.gzzr7300_45V1V3_R2.fastq.gz


F7300.S046Subject8.Visit3.Szr7300_46V1V3_R1.fastq.gzzr7300_46V1V3_R2.fastq.gz


F7300.S047Subject9.Visit3.Szr7300_47V1V3_R1.fastq.gzzr7300_47V1V3_R2.fastq.gz


F7300.S048Subject10.Visit3.Szr7300_48V1V3_R1.fastq.gzzr7300_48V1V3_R2.fastq.gz


F7300.S049Subject11.Visit3.Szr7300_49V1V3_R1.fastq.gzzr7300_49V1V3_R2.fastq.gz


F7300.S004Subject4.Visit1.Szr7300_4V1V3_R1.fastq.gzzr7300_4V1V3_R2.fastq.gz


F7300.S050Subject12.Visit3.Szr7300_50V1V3_R1.fastq.gzzr7300_50V1V3_R2.fastq.gz


F7300.S051Subject13.Visit3.Szr7300_51V1V3_R1.fastq.gzzr7300_51V1V3_R2.fastq.gz


F7300.S052Subject15.Visit3.Szr7300_52V1V3_R1.fastq.gzzr7300_52V1V3_R2.fastq.gz


F7300.S053Subject16.Visit3.Szr7300_53V1V3_R1.fastq.gzzr7300_53V1V3_R2.fastq.gz


F7300.S054Subject17.Visit3.Szr7300_54V1V3_R1.fastq.gzzr7300_54V1V3_R2.fastq.gz


F7300.S055Subject18.Visit3.Szr7300_55V1V3_R1.fastq.gzzr7300_55V1V3_R2.fastq.gz


F7300.S056Subject19.Visit3.Szr7300_56V1V3_R1.fastq.gzzr7300_56V1V3_R2.fastq.gz


F7300.S057Subject20.Visit3.Szr7300_57V1V3_R1.fastq.gzzr7300_57V1V3_R2.fastq.gz


F7300.S058Subject1.Visit1.Pzr7300_58V1V3_R1.fastq.gzzr7300_58V1V3_R2.fastq.gz


F7300.S059Subject2.Visit1.Pzr7300_59V1V3_R1.fastq.gzzr7300_59V1V3_R2.fastq.gz


F7300.S005Subject5.Visit1.Szr7300_5V1V3_R1.fastq.gzzr7300_5V1V3_R2.fastq.gz


F7300.S060Subject3.Visit1.Pzr7300_60V1V3_R1.fastq.gzzr7300_60V1V3_R2.fastq.gz


F7300.S061Subject4.Visit1.Pzr7300_61V1V3_R1.fastq.gzzr7300_61V1V3_R2.fastq.gz


F7300.S062Subject5.Visit1.Pzr7300_62V1V3_R1.fastq.gzzr7300_62V1V3_R2.fastq.gz


F7300.S063Subject6.Visit1.Pzr7300_63V1V3_R1.fastq.gzzr7300_63V1V3_R2.fastq.gz


F7300.S064Subject7.Visit1.Pzr7300_64V1V3_R1.fastq.gzzr7300_64V1V3_R2.fastq.gz


F7300.S065Subject8.Visit1.Pzr7300_65V1V3_R1.fastq.gzzr7300_65V1V3_R2.fastq.gz


F7300.S066Subject9.Visit1.Pzr7300_66V1V3_R1.fastq.gzzr7300_66V1V3_R2.fastq.gz


F7300.S067Subject10.Visit1.Pzr7300_67V1V3_R1.fastq.gzzr7300_67V1V3_R2.fastq.gz


F7300.S068Subject11.Visit1.Pzr7300_68V1V3_R1.fastq.gzzr7300_68V1V3_R2.fastq.gz


F7300.S069Subject12.Visit1.Pzr7300_69V1V3_R1.fastq.gzzr7300_69V1V3_R2.fastq.gz


F7300.S006Subject6.Visit1.Szr7300_6V1V3_R1.fastq.gzzr7300_6V1V3_R2.fastq.gz


F7300.S070Subject13.Visit1.Pzr7300_70V1V3_R1.fastq.gzzr7300_70V1V3_R2.fastq.gz


F7300.S071Subject15.Visit1.Pzr7300_71V1V3_R1.fastq.gzzr7300_71V1V3_R2.fastq.gz


F7300.S072Subject16.Visit1.Pzr7300_72V1V3_R1.fastq.gzzr7300_72V1V3_R2.fastq.gz


F7300.S073Subject17.Visit1.Pzr7300_73V1V3_R1.fastq.gzzr7300_73V1V3_R2.fastq.gz


F7300.S074Subject18.Visit1.Pzr7300_74V1V3_R1.fastq.gzzr7300_74V1V3_R2.fastq.gz


F7300.S075Subject19.Visit1.Pzr7300_75V1V3_R1.fastq.gzzr7300_75V1V3_R2.fastq.gz


F7300.S076Subject20.Visit1.Pzr7300_76V1V3_R1.fastq.gzzr7300_76V1V3_R2.fastq.gz


F7300.S077Subject1.Visit2.Pzr7300_77V1V3_R1.fastq.gzzr7300_77V1V3_R2.fastq.gz


F7300.S078Subject2.Visit2.Pzr7300_78V1V3_R1.fastq.gzzr7300_78V1V3_R2.fastq.gz


F7300.S079Subject3.Visit2.Pzr7300_79V1V3_R1.fastq.gzzr7300_79V1V3_R2.fastq.gz


F7300.S007Subject7.Visit1.Szr7300_7V1V3_R1.fastq.gzzr7300_7V1V3_R2.fastq.gz


F7300.S080Subject4.Visit2.Pzr7300_80V1V3_R1.fastq.gzzr7300_80V1V3_R2.fastq.gz


F7300.S081Subject5.Visit2.Pzr7300_81V1V3_R1.fastq.gzzr7300_81V1V3_R2.fastq.gz


F7300.S082Subject6.Visit2.Pzr7300_82V1V3_R1.fastq.gzzr7300_82V1V3_R2.fastq.gz


F7300.S083Subject7.Visit2.Pzr7300_83V1V3_R1.fastq.gzzr7300_83V1V3_R2.fastq.gz


F7300.S084Subject8.Visit2.Pzr7300_84V1V3_R1.fastq.gzzr7300_84V1V3_R2.fastq.gz


F7300.S085Subject9.Visit2.Pzr7300_85V1V3_R1.fastq.gzzr7300_85V1V3_R2.fastq.gz


F7300.S086Subject10.Visit2.Pzr7300_86V1V3_R1.fastq.gzzr7300_86V1V3_R2.fastq.gz


F7300.S087Subject11.Visit2.Pzr7300_87V1V3_R1.fastq.gzzr7300_87V1V3_R2.fastq.gz


F7300.S088Subject12.Visit2.Pzr7300_88V1V3_R1.fastq.gzzr7300_88V1V3_R2.fastq.gz


F7300.S089Subject13.Visit2.Pzr7300_89V1V3_R1.fastq.gzzr7300_89V1V3_R2.fastq.gz


F7300.S008Subject8.Visit1.Szr7300_8V1V3_R1.fastq.gzzr7300_8V1V3_R2.fastq.gz


F7300.S090Subject15.Visit2.Pzr7300_90V1V3_R1.fastq.gzzr7300_90V1V3_R2.fastq.gz


F7300.S091Subject16.Visit2.Pzr7300_91V1V3_R1.fastq.gzzr7300_91V1V3_R2.fastq.gz


F7300.S092Subject17.Visit2.Pzr7300_92V1V3_R1.fastq.gzzr7300_92V1V3_R2.fastq.gz


F7300.S093Subject18.Visit2.Pzr7300_93V1V3_R1.fastq.gzzr7300_93V1V3_R2.fastq.gz


F7300.S094Subject19.Visit2.Pzr7300_94V1V3_R1.fastq.gzzr7300_94V1V3_R2.fastq.gz


F7300.S095Subject20.Visit2.Pzr7300_95V1V3_R1.fastq.gzzr7300_95V1V3_R2.fastq.gz


F7300.S096Subject1.Visit3.Pzr7300_96V1V3_R1.fastq.gzzr7300_96V1V3_R2.fastq.gz


F7300.S097Subject2.Visit3.Pzr7300_97V1V3_R1.fastq.gzzr7300_97V1V3_R2.fastq.gz


F7300.S098Subject3.Visit3.Pzr7300_98V1V3_R1.fastq.gzzr7300_98V1V3_R2.fastq.gz


F7300.S099Subject4.Visit3.Pzr7300_99V1V3_R1.fastq.gzzr7300_99V1V3_R2.fastq.gz


F7300.S009Subject9.Visit1.Szr7300_9V1V3_R1.fastq.gzzr7300_9V1V3_R2.fastq.gz

Please download and save the file to your computer storage device. The download link will expire after 60 days upon your receiving of this report.

Raw sequence data download link:

 

VI. Analysis - DADA2 Read Processing

What is DADA2?

DADA2 is a software package that models and corrects Illumina-sequenced amplicon errors. DADA2 infers sample sequences exactly, without coarse-graining into OTUs, and resolves differences of as little as one nucleotide. DADA2 identified more real variants and output fewer spurious sequences than other methods.

DADA2’s advantage is that it uses more of the data. The DADA2 error model incorporates quality information, which is ignored by all other methods after filtering. The DADA2 error model incorporates quantitative abundances, whereas most other methods use abundance ranks if they use abundance at all. The DADA2 error model identifies the differences between sequences, eg. A->C, whereas other methods merely count the mismatches. DADA2 can parameterize its error model from the data itself, rather than relying on previous datasets that may or may not reflect the PCR and sequencing protocols used in your study.

DADA2 Publication: Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016 Jul;13(7):581-3. doi: 10.1038/nmeth.3869. Epub 2016 May 23. PMID: 27214047; PMCID: PMC4927377.

DADA2 Software Package is available as an R package at : https://benjjneb.github.io/dada2/index.html

Analysis Procedures:

DADA2 pipeline includes several tools for read quality control, including quality filtering, trimming, denoising, pair merging and chimera filtering. Below are the major processing steps of DADA2:

Step 1. Read trimming based on sequence quality The quality of NGS Illumina sequences often decreases toward the end of the reads. DADA2 allows to trim off the poor quality read ends in order to improve the error model building and pair mergicing performance.

Step 2. Learn the Error Rates The DADA2 algorithm makes use of a parametric error model (err) and every amplicon dataset has a different set of error rates. The learnErrors method learns this error model from the data, by alternating estimation of the error rates and inference of sample composition until they converge on a jointly consistent solution. As in many machine-learning problems, the algorithm must begin with an initial guess, for which the maximum possible error rates in this data are used (the error rates if only the most abundant sequence is correct and all the rest are errors).

Step 3. Infer amplicon sequence variants (ASVs) based on the error model built in previous step. This step is also called sequence "denoising". The outcome of this step is a list of ASVs that are the equivalent of oligonucleotides.

Step 4. Merge paired reads. If the sequencing products are read pairs, DADA2 will merge the R1 and R2 ASVs into single sequences. Merging is performed by aligning the denoised forward reads with the reverse-complement of the corresponding denoised reverse reads, and then constructing the merged “contig” sequences. By default, merged sequences are only output if the forward and reverse reads overlap by at least 12 bases, and are identical to each other in the overlap region (but these conditions can be changed via function arguments).

Step 5. Remove chimera. The core dada method corrects substitution and indel errors, but chimeras remain. Fortunately, the accuracy of sequence variants after denoising makes identifying chimeric ASVs simpler than when dealing with fuzzy OTUs. Chimeric sequences are identified if they can be exactly reconstructed by combining a left-segment and a right-segment from two more abundant “parent” sequences. The frequency of chimeric sequences varies substantially from dataset to dataset, and depends on on factors including experimental procedures and sample complexity.

Results

1. Read Quality Plots NGS sequence analaysis starts with visualizing the quality of the sequencing. Below are the quality plots of the first sample for the R1 and R2 reads separately. In gray-scale is a heat map of the frequency of each quality score at each base position. The mean quality score at each position is shown by the green line, and the quartiles of the quality score distribution by the orange lines. The forward reads are usually of better quality. It is a common practice to trim the last few nucleotides to avoid less well-controlled errors that can arise there. The trimming affects the downstream steps including error model building, merging and chimera calling. FOMC uses an empirical approach to test many combinations of different trim length in order to achieve best final amplicon sequence variants (ASVs), see the next section “Optimal trim length for ASVs”.

Quality plots for all samples:

2. Optimal trim length for ASVs The final number of merged and chimera-filtered ASVs depends on the quality filtering (hence trimming) in the very beginning of the DADA2 pipeline. In order to achieve highest number of ASVs, an empirical approach was used -

  1. Create a random subset of each sample consisting of 5,000 R1 and 5,000 R2 (to reduce computation time)
  2. Trim 10 bases at a time from the ends of both R1 and R2 up to 50 bases
  3. For each combination of trimmed length (e.g., 300x300, 300x290, 290x290 etc), the trimmed reads are subject to the entire DADA2 pipeline for chimera-filtered merged ASVs
  4. The combination with highest percentage of the input reads becoming final ASVs is selected for the complete set of data

Below is the result of such operation, showing ASV percentages of total reads for all trimming combinations (1st Column = R1 lengths in bases; 1st Row = R2 lengths in bases):

R1/R2281271261251241231
3219.10%26.48%34.67%40.39%41.56%37.64%
3119.60%27.93%37.87%45.39%42.18%24.08%
3019.91%28.55%38.49%40.80%24.15%13.27%
2919.93%28.83%33.79%22.17%13.22%9.38%
28110.15%25.34%17.23%12.28%9.10%4.31%
2719.90%12.30%9.59%8.39%4.03%1.92%

Based on the above result, the trim length combination of R1 = 311 bases and R2 = 251 bases (highlighted red above), was chosen for generating final ASVs for all sequences. This combination generated highest number of merged non-chimeric ASVs and was used for downstream analyses, if requested.

3. Error plots from learning the error rates After DADA2 building the error model for the set of data, it is always worthwhile, as a sanity check if nothing else, to visualize the estimated error rates. The error rates for each possible transition (A→C, A→G, …) are shown below. Points are the observed error rates for each consensus quality score. The black line shows the estimated error rates after convergence of the machine-learning algorithm. The red line shows the error rates expected under the nominal definition of the Q-score. The ideal result would be the estimated error rates (black line) are a good fit to the observed rates (points), and the error rates drop with increased quality as expected.

Forward Read R1 Error Plot


Reverse Read R2 Error Plot

The PDF version of these plots are available here:

 

4. DADA2 Result Summary The table below shows the summary of the DADA2 analysis, tracking paired read counts of each samples for all the steps during DADA2 denoising process - including end-trimming (filtered), denoising (denoisedF, denoisedF), pair merging (merged) and chimera removal (nonchim).

Sample IDF7300.S001F7300.S002F7300.S003F7300.S004F7300.S005F7300.S006F7300.S007F7300.S008F7300.S009F7300.S010F7300.S011F7300.S012F7300.S013F7300.S014F7300.S015F7300.S016F7300.S017F7300.S018F7300.S019F7300.S020F7300.S021F7300.S022F7300.S023F7300.S024F7300.S025F7300.S026F7300.S027F7300.S028F7300.S029F7300.S030F7300.S031F7300.S032F7300.S033F7300.S034F7300.S035F7300.S036F7300.S037F7300.S038F7300.S039F7300.S040F7300.S041F7300.S042F7300.S043F7300.S044F7300.S045F7300.S046F7300.S047F7300.S048F7300.S049F7300.S050F7300.S051F7300.S052F7300.S053F7300.S054F7300.S055F7300.S056F7300.S057F7300.S058F7300.S059F7300.S060F7300.S061F7300.S062F7300.S063F7300.S064F7300.S065F7300.S066F7300.S067F7300.S068F7300.S069F7300.S070F7300.S071F7300.S072F7300.S073F7300.S074F7300.S075F7300.S076F7300.S077F7300.S078F7300.S079F7300.S080F7300.S081F7300.S082F7300.S083F7300.S084F7300.S085F7300.S086F7300.S087F7300.S088F7300.S089F7300.S090F7300.S091F7300.S092F7300.S093F7300.S094F7300.S095F7300.S096F7300.S097F7300.S098F7300.S099F7300.S100F7300.S101F7300.S102F7300.S103F7300.S104F7300.S105F7300.S106F7300.S107F7300.S108F7300.S109F7300.S110F7300.S111F7300.S112F7300.S113F7300.S114Row SumPercentage
input32,78732,60836,03720,60450,47935,90933,39936,92433,39934,80726,72730,19226,02124,62728,41331,43434,68935,54331,82829,53435,28238,85236,91639,05137,85126,93729,93832,41027,92129,78528,81638,41232,27627,48233,45836,41825,20331,51929,60235,29640,87528,77025,70326,92233,09938,51733,47439,54736,14330,74028,75428,88031,12329,74832,57132,17332,02824,23327,89031,52133,60128,99528,41637,37736,27628,41232,95425,28728,53037,16230,58634,63531,61432,55823,41825,38425,68526,81534,59029,99034,14037,49731,18130,20331,54224,11335,98734,23033,75831,97428,13531,63923,82224,86728,47424,83529,19726,83225,77223,57532,11029,00530,51227,59728,30427,42334,14025,44829,18132,30433,92429,97828,31829,8263,554,225100.00%
filtered32,76232,59436,01620,58550,43335,89233,37836,90933,38534,78126,70430,17626,00424,60828,39231,42334,66635,52731,80129,51335,25538,82936,88739,00937,83426,91029,91032,38727,90529,77228,78538,39532,25927,45233,44536,39825,18131,49729,58435,27840,84428,75525,67126,89233,06938,49233,45239,52836,11930,71828,73728,86131,09929,73132,53432,14932,00624,21827,86331,50233,57928,97528,40137,35536,25628,39332,92625,24128,49937,13230,56034,61731,59332,53323,40925,37225,66926,79134,54729,97134,11637,46731,16530,18531,52924,08235,95934,20033,73731,95428,11831,57423,80324,85428,45424,81829,17926,80725,74923,55332,08828,98230,48627,58928,28027,39534,10625,43229,17332,27833,91329,95728,30429,8013,551,66799.93%
denoisedF30,75131,04633,55119,23448,26333,53931,12934,94931,47632,43324,68827,56823,82523,28826,50829,12032,37233,81829,78527,92433,15436,50934,99536,63135,22025,17728,19030,32925,86827,90226,54536,26530,41725,62231,79734,73623,43729,64527,73033,29337,96927,12823,95424,90631,03936,23231,60737,30933,54928,91726,66227,17128,94627,64230,14330,33030,05222,68726,22129,80331,36127,18626,82535,03734,11426,82230,70423,26626,32834,37628,36532,79428,99430,42222,15224,17723,80025,19032,61128,15232,11235,28529,19028,26629,74422,38533,44031,74231,81329,81526,36929,10922,46623,30327,02223,01727,36624,98924,33921,54530,15827,17828,25026,08326,33125,50031,57723,59327,45930,44131,77328,00726,81227,6563,327,80793.63%
denoisedR30,33130,28433,31417,48547,59532,88830,92534,67330,95331,29024,61927,61223,47822,20526,19628,40631,96233,09529,35227,59732,74635,72834,33635,99634,49124,37627,78929,93125,12227,43626,45635,68129,84925,02730,88834,15923,19229,12227,18432,77937,57326,27223,69624,71330,33035,64530,63636,71132,77828,35826,45526,44728,63027,32729,95629,42029,50222,32125,39828,72728,22126,26826,29634,03533,42826,39230,50322,90424,23634,11627,30630,53928,55730,02521,08823,75123,87724,36430,31626,41931,29734,10228,71627,46329,28422,23832,67929,82531,30129,00025,26329,26721,20922,62626,24222,87026,61724,24522,66320,95329,47626,45027,85024,93925,37324,67130,65622,69726,87229,74330,86527,03125,80426,8023,247,17391.36%
merged25,06726,62628,89914,88441,27027,68925,98929,58626,59926,19020,37222,62719,48118,42921,70622,80626,47429,05225,33024,46628,90230,12929,37730,25728,70420,94123,76826,01320,89822,18221,83329,96025,19821,07326,66329,12819,45724,38922,39328,16231,26022,84620,04520,53725,49429,80625,79530,56927,01624,64622,00721,82122,96222,25624,45622,83925,67716,52120,87524,33121,98620,27621,49527,46026,70922,90725,04618,75118,82426,25621,64024,57722,05622,54315,92821,09518,22520,53624,36119,87525,28127,06322,51923,47526,03816,75726,06922,14527,15823,27720,47323,30717,99017,35522,40718,40922,11419,35418,69015,10923,21021,91722,79421,85219,27418,15222,28016,40822,53725,24225,29121,27821,23322,0282,673,76075.23%
nonchim13,80014,27914,2609,49918,83116,81214,29614,92314,17514,93410,28511,71710,83210,72211,31612,45914,10616,76614,83414,84615,07316,55615,82014,84218,18113,82114,85714,65812,01811,30711,88815,58011,79211,27918,47019,45211,00712,66911,92814,80916,67815,22010,89413,60813,10016,43014,79816,17515,02512,01512,45911,67512,42212,88513,13813,08515,1628,71411,33413,43913,22012,01911,57415,44515,6869,59714,86211,21512,69711,84711,94617,64113,99313,3489,71812,7658,63311,06113,79111,57112,68416,91312,41913,44111,56210,95714,47312,38011,07611,18913,31113,19111,7889,26413,3588,21711,51712,97910,8688,45014,62512,81713,14512,54511,77310,81113,1569,07610,27712,96914,40314,41710,17711,3201,492,13241.98%

This table can be downloaded as an Excel table below:

 

5. DADA2 Amplicon Sequence Variants (ASVs). A total of 16272 unique merged and chimera-free ASV sequences were identified, and their corresponding read counts for each sample are available in the "ASV Read Count Table" with rows for the ASV sequences and columns for sample. This read count table can be used for microbial profile comparison among different samples and the sequences provided in the table can be used to taxonomy assignment.

 

The table can be downloaded from this link:

 
 

Sample Meta Information

#SampleIDSample NameSourceVisitGroup
F7300.S001Subject1.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S002Subject2.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S003Subject3.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S004Subject4.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S005Subject5.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S006Subject6.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S007Subject7.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S008Subject8.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S009Subject9.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S010Subject10.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S011Subject11.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S012Subject12.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S013Subject13.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S014Subject15.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S015Subject16.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S016Subject17.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S017Subject18.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S018Subject19.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S019Subject20.Visit1.SHuman salivaVisit1Saliva Visit 1
F7300.S020Subject1.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S021Subject2.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S022Subject3.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S023Subject4.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S024Subject5.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S025Subject6.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S026Subject7.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S027Subject8.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S028Subject9.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S029Subject10.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S030Subject11.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S031Subject12.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S032Subject13.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S033Subject15.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S034Subject16.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S035Subject17.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S036Subject18.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S037Subject19.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S038Subject20.Visit2.SHuman salivaVisit2Saliva Visit 2
F7300.S039Subject1.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S040Subject2.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S041Subject3.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S042Subject4.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S043Subject5.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S044Subject6.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S045Subject7.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S046Subject8.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S047Subject9.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S048Subject10.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S049Subject11.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S050Subject12.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S051Subject13.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S052Subject15.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S053Subject16.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S054Subject17.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S055Subject18.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S056Subject19.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S057Subject20.Visit3.SHuman salivaVisit3Saliva Visit 3
F7300.S058Subject1.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S059Subject2.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S060Subject3.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S061Subject4.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S062Subject5.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S063Subject6.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S064Subject7.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S065Subject8.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S066Subject9.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S067Subject10.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S068Subject11.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S069Subject12.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S070Subject13.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S071Subject15.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S072Subject16.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S073Subject17.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S074Subject18.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S075Subject19.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S076Subject20.Visit1.PHuman plaqueVisit1Plaque Visit 1
F7300.S077Subject1.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S078Subject2.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S079Subject3.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S080Subject4.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S081Subject5.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S082Subject6.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S083Subject7.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S084Subject8.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S085Subject9.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S086Subject10.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S087Subject11.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S088Subject12.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S089Subject13.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S090Subject15.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S091Subject16.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S092Subject17.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S093Subject18.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S094Subject19.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S095Subject20.Visit2.PHuman plaqueVisit2Plaque Visit 2
F7300.S096Subject1.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S097Subject2.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S098Subject3.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S099Subject4.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S100Subject5.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S101Subject6.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S102Subject7.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S103Subject8.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S104Subject9.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S105Subject10.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S106Subject11.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S107Subject12.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S108Subject13.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S109Subject15.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S110Subject16.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S111Subject17.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S112Subject18.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S113Subject19.Visit3.PHuman plaqueVisit3Plaque Visit 3
F7300.S114Subject20.Visit3.PHuman plaqueVisit3Plaque Visit 3
 
 

ASV Read Counts by Samples

#Sample IDRead Count
F7300.S0968,217
F7300.S1008,450
F7300.S0778,633
F7300.S0588,714
F7300.S1089,076
F7300.S0949,264
F7300.S0049,499
F7300.S0669,597
F7300.S0759,718
F7300.S11310,177
F7300.S10910,277
F7300.S01110,285
F7300.S01410,722
F7300.S10610,811
F7300.S01310,832
F7300.S09910,868
F7300.S04310,894
F7300.S08610,957
F7300.S03711,007
F7300.S07811,061
F7300.S08911,076
F7300.S09011,189
F7300.S06811,215
F7300.S03411,279
F7300.S03011,307
F7300.S01511,316
F7300.S11411,320
F7300.S05911,334
F7300.S09711,517
F7300.S08511,562
F7300.S08011,571
F7300.S06311,574
F7300.S05211,675
F7300.S01211,717
F7300.S10511,773
F7300.S09311,788
F7300.S03311,792
F7300.S07011,847
F7300.S03111,888
F7300.S03911,928
F7300.S07111,946
F7300.S05012,015
F7300.S02912,018
F7300.S06212,019
F7300.S08812,380
F7300.S08312,419
F7300.S05312,422
F7300.S01612,459
F7300.S05112,459
F7300.S10412,545
F7300.S03812,669
F7300.S08112,684
F7300.S06912,697
F7300.S07612,765
F7300.S10212,817
F7300.S05412,885
F7300.S11012,969
F7300.S09812,979
F7300.S05613,085
F7300.S04513,100
F7300.S05513,138
F7300.S10313,145
F7300.S10713,156
F7300.S09213,191
F7300.S06113,220
F7300.S09113,311
F7300.S07413,348
F7300.S09513,358
F7300.S06013,439
F7300.S08413,441
F7300.S04413,608
F7300.S07913,791
F7300.S00113,800
F7300.S02613,821
F7300.S07313,993
F7300.S01714,106
F7300.S00914,175
F7300.S00314,260
F7300.S00214,279
F7300.S00714,296
F7300.S11114,403
F7300.S11214,417
F7300.S08714,473
F7300.S10114,625
F7300.S02814,658
F7300.S04714,798
F7300.S04014,809
F7300.S01914,834
F7300.S02414,842
F7300.S02014,846
F7300.S02714,857
F7300.S06714,862
F7300.S00814,923
F7300.S01014,934
F7300.S04915,025
F7300.S02115,073
F7300.S05715,162
F7300.S04215,220
F7300.S06415,445
F7300.S03215,580
F7300.S06515,686
F7300.S02315,820
F7300.S04816,175
F7300.S04616,430
F7300.S02216,556
F7300.S04116,678
F7300.S01816,766
F7300.S00616,812
F7300.S08216,913
F7300.S07217,641
F7300.S02518,181
F7300.S03518,470
F7300.S00518,831
F7300.S03619,452
 
 
 

VII. Analysis - Read Taxonomy Assignment

Read Taxonomy Assignment - Methods

 

The species-level, open-reference 16S rRNA NGS reads taxonomy assignment pipeline

Version 20210310
 

1. Raw sequences reads in FASTA format were BLASTN-searched against a combined set of 16S rRNA reference sequences. It consists of MOMD (version 0.1), the HOMD (version 15.2 http://www.homd.org/index.php?name=seqDownload&file&type=R ), HOMD 16S rRNA RefSeq Extended Version 1.1 (EXT), GreenGene Gold (GG) (http://greengenes.lbl.gov/Download/Sequence_Data/Fasta_data_files/gold_strains_gg16S_aligned.fasta.gz) , and the NCBI 16S rRNA reference sequence set (https://ftp.ncbi.nlm.nih.gov/blast/db/16S_ribosomal_RNA.tar.gz). These sequences were screened and combined to remove short sequences (<1000nt), chimera, duplicated and sub-sequences, as well as sequences with poor taxonomy annotation (e.g., without species information). This process resulted in 1,015 from HOMD V15.22, 495 from EXT, 3,940 from GG and 18,044 from NCBI, a total of 25,120 sequences. Altogether these sequence represent a total of 15,601 oral and non-oral microbial species.

The NCBI BLASTN version 2.7.1+ (Zhang et al, 2000) was used with the default parameters. Reads with ≥ 98% sequence identity to the matched reference and ≥ 90% alignment length (i.e., ≥ 90% of the read length that was aligned to the reference and was used to calculate the sequence percent identity) were classified based on the taxonomy of the reference sequence with highest sequence identity. If a read matched with reference sequences representing more than one species with equal percent identity and alignment length, it was subject to chimera checking with USEARCH program version v8.1.1861 (Edgar 2010). Non-chimeric reads with multi-species best hits were considered valid and were assigned with a unique species notation (e.g., spp) denoting unresolvable multiple species.

2. Unassigned reads (i.e., reads with < 98% identity or < 90% alignment length) were pooled together and reads < 200 bases were removed. The remaining reads were subject to the de novo operational taxonomy unit (OTU) calling and chimera checking using the USEARCH program version v8.1.1861 (Edgar 2010). The de novo OTU calling and chimera checking was done using 98% as the sequence identity cutoff, i.e., the species-level OTU. The output of this step produced species-level de novo clustered OTUs with 98% identity. Representative reads from each of the OTUs/species were then BLASTN-searched against the same reference sequence set again to determine the closest species for these potential novel species. These potential novel species were pooled together with the reads that were signed to specie-level in the previous step, for down-stream analyses.

Reference:
Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010 Oct 1;26(19):2460-1. doi: 10.1093/bioinformatics/btq461. Epub 2010 Aug 12. PubMed PMID: 20709691.

3. Designations used in the taxonomy:

	1) Taxonomy levels are indicated by these prefixes:
	
	   k__: domain/kingdom
	   p__: phylum
	   c__: class
	   o__: order
	   f__: family
	   g__: genus  
	   s__: species
	
	   Example: 
	
	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Blautia;s__faecis
		
	2) Unique level identified – known species:
	   
	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Roseburia;s__hominis
	
	   The above example shows some reads match to a single species (all levels are unique)
	
	3) Non-unique level identified – known species:

	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Roseburia;s__multispecies_spp123_3
	   
	   The above example “s__multispecies_spp123_3” indicates certain reads equally match to 3 species of the 
	   genus Roseburia; the “spp123” is a temporally assigned species ID.
	
	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__multigenus;s__multispecies_spp234_5
	   
	   The above example indicates certain reads match equally to 5 different species, which belong to multiple genera.; 
	   the “spp234” is a temporally assigned species ID.
	
	4) Unique level identified – unknown species, potential novel species:
	   
	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Roseburia;s__ hominis_nov_97%
	   
	   The above example indicates that some reads have no match to any of the reference sequences with 
	   sequence identity ≥ 98% and percent coverage (alignment length)  ≥ 98% as well. However this groups 
	   of reads (actually the representative read from a de novo  OTU) has 96% percent identity to 
	   Roseburia hominis, thus this is a potential novel species, closest to Roseburia hominis. 
	   (But they are not the same species).
	
	5) Multiple level identified – unknown species, potential novel species:
	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Roseburia;s__ multispecies_sppn123_3_nov_96%
	
	   The above example indicates that some reads have no match to any of the reference sequences 
	   with sequence identity ≥ 98% and percent coverage (alignment length)  ≥ 98% as well. 
	   However this groups of reads (actually the representative read from a de novo  OTU) 
	   has 96% percent identity equally to 3 species in Roseburia. Thus this is no single 
	   closest species, instead this group of reads match equally to multiple species at 96%. 
	   Since they have passed chimera check so they represent a novel species. “sppn123” is a 
	   temporary ID for this potential novel species.

 
4. The taxonomy assignment algorithm is illustrated in this flow char below:
 
 
 
 

Read Taxonomy Assignment - Result Summary *

CodeCategoryMPC=0% (>=1 read)MPC=0.01%(>=148 reads)
ATotal reads1,492,1321,492,132
BTotal assigned reads1,482,8251,482,825
CAssigned reads in species with read count < MPC011,770
DAssigned reads in samples with read count < 50000
ETotal samples114114
FSamples with reads >= 500114114
GSamples with reads < 50000
HTotal assigned reads used for analysis (B-C-D)1,482,8251,471,055
IReads assigned to single species1,425,4881,419,919
JReads assigned to multiple species32,37831,163
KReads assigned to novel species24,95919,973
LTotal number of species570308
MNumber of single species345255
NNumber of multi-species3615
ONumber of novel species18938
PTotal unassigned reads9,3079,307
QChimeric reads953953
RReads without BLASTN hits55
SOthers: short, low quality, singletons, etc.8,3498,349
A=B+P=C+D+H+Q+R+S
E=F+G
B=C+D+H
H=I+J+K
L=M+N+O
P=Q+R+S
* MPC = Minimal percent (of all assigned reads) read count per species, species with read count < MPC were removed.
* Samples with reads < 500 were removed from downstream analyses.
* The assignment result from MPC=0.1% was used in the downstream analyses.
 
 
 

Read Taxonomy Assignment - ASV Species-Level Read Counts Table

This table shows the read counts for each sample (columns) and each species identified based on the ASV sequences. The downstream analyses were based on this table.
SPIDTaxonomyF7300.S001F7300.S002F7300.S003F7300.S004F7300.S005F7300.S006F7300.S007F7300.S008F7300.S009F7300.S010F7300.S011F7300.S012F7300.S013F7300.S014F7300.S015F7300.S016F7300.S017F7300.S018F7300.S019F7300.S020F7300.S021F7300.S022F7300.S023F7300.S024F7300.S025F7300.S026F7300.S027F7300.S028F7300.S029F7300.S030F7300.S031F7300.S032F7300.S033F7300.S034F7300.S035F7300.S036F7300.S037F7300.S038F7300.S039F7300.S040F7300.S041F7300.S042F7300.S043F7300.S044F7300.S045F7300.S046F7300.S047F7300.S048F7300.S049F7300.S050F7300.S051F7300.S052F7300.S053F7300.S054F7300.S055F7300.S056F7300.S057F7300.S058F7300.S059F7300.S060F7300.S061F7300.S062F7300.S063F7300.S064F7300.S065F7300.S066F7300.S067F7300.S068F7300.S069F7300.S070F7300.S071F7300.S072F7300.S073F7300.S074F7300.S075F7300.S076F7300.S077F7300.S078F7300.S079F7300.S080F7300.S081F7300.S082F7300.S083F7300.S084F7300.S085F7300.S086F7300.S087F7300.S088F7300.S089F7300.S090F7300.S091F7300.S092F7300.S093F7300.S094F7300.S095F7300.S096F7300.S097F7300.S098F7300.S099F7300.S100F7300.S101F7300.S102F7300.S103F7300.S104F7300.S105F7300.S106F7300.S107F7300.S108F7300.S109F7300.S110F7300.S111F7300.S112F7300.S113F7300.S114
SP1Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella;parvula36853160812756036455457437948313556143849377248662230130027839659829750834169275876526434058045562297340529728336442146297967239754055071871134559164248758069936539915963663824911537542863496231361013420843829158739370518410532473115982139397989282651009572744279254112517237238846984414367614300935122086434021736592209791757297525345400855688340
SP10Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT1710640000000000000000000140000000000000000005203000000360000000230106910000750141103068009201240201362900002435610767440006125125003480000200411251360000001800
SP100Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;sputigena0000026000230000069000000002600000003404700000000350003000000000002754000069014900000122008108200018606806602082200186001013725000970002650002259122000
SP101Bacteria;Firmicutes;Bacilli;Bacillales;Gemellaceae;Gemella;haemolysans01132159865176523373326171201130320846012999003935718034588001930336144001161081630084000075072235822048094031959041000000000006000000000000800000000000039000065000610000002500000
SP102Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;artemidis000000000000000000000000000000000000000000000000000000000189130000200000023000880076000714900000110000000680000150000003800007000
SP103Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._Oral_Taxon_71200029332300000000000001900210340000001900000016000049000000000000018900170010307800005000012900141023594000000004590003812201330023082000034011400021700
SP104Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;oralis_subsp._dentisani_clade_05800530002100103000650000000560004001160016000000002100060007600702421000000320035000119071925000000014000000129003901600000072000250012900911460000
SP105Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;intermedius00332020001300120000400005005014140316422001370000000431617098100000000001566001815701013186490141260225090181559615518483140021739164140160560543151431444637101753235052890700266
SP106Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp. HMT0640000005300000000001800240250000000000000000000001700000000000000000014100000000001010010403500000000000002000483000069000000000000
SP107Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._str._C150000000000000000003396000000000000000000230200000000000000000059050000000000000000001053000000000000000000310000000000000000006480
SP108Bacteria;Firmicutes;Clostridia;Eubacteriales;Eubacteriales Family XIII. Incertae Sedis;Mogibacterium;vescum812506740680337752437309760015300004151010056602926385001634747180015806951476445058083045108756899012800000000000047000570000000000000000000000000000100000042007400
SP109Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-1];bacterium HMT3480000041000000320000000000000000000000000000000000000000000000000672024301631370102330547163000000084082401258910400508524150000000550327016015812415600011300
SP11Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;subflava025000019800317000000001600000015200524000000000000000237004010040000000360000000011387000012200019000292742000000000000001500045600004840006700
SP110Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;parasanguinis_I2012914214022520537153101017001542404803931112502141812034014519504008641780120146226371021501911159387176046127195821775019300000000000000000001900001201900001330000000002200423200000000000
SP112Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Aggregatibacter;sp. HMT458000000000000172000000001310000000000000000008900000000000000000000000000165414700045000000000000666000042510000196000022100030221000000
SP113Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp. HMT06119706342000128024001500299846000060510100010000008293110135150007236617000027002134208813417410055000000000000000000000002500000000000000000070000010000000000000
SP114Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-9];[Eubacterium]_brachy000000000000260000530004700160000000000031000000000520003400000000019005333000430050190150000410011200014720012605032000000000220900149053853413241
SP115Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Stomatobaculum;sp. HMT097217000001225100470008200049000126500134000000820000158157001140010915264000069116011501060002300000000000000000034000000000000000580045000000000000000
SP116Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._str._2136FAA0055041000000000000153006002500000001610005000000000000000000002059002410930000000001806401963701350870000000935012020943293710840000000000000090
SP117Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;granulosa000003500000000000000000224400000000000000000076000000000000000000139000145902400089000000070014200007160270480000000168000850106780005000
SP118Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-1];bacterium HMT35245224352002161137129129680003616502614812341040256777791259028000420039400137136002514209323722360090004900013000000000000000000800000000000000000000320000000000000
SP119Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-1];bacterium HMT34700000000000160000000000000000000000000000000000000000000000000000023405100000000000007500313147000007601040000000009000270000000
SP12Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Corynebacteriaceae;Corynebacterium;matruchotii32000000000001790000000026500000840005600000000000000540001290007004481413805695281251035013847644797214718191421360006074521635307989821090147148059527828878581596415220805457913492670404337704316170311117000446095
SP120Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Fretibacterium;sp. HMT35900000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000017700000000000000000
SP121Bacteria;Actinobacteria;Actinomycetia;Bifidobacteriales;Bifidobacteriaceae;Scardovia;wiggsiae00000000000000000350000231180000000000000000005900000000000016100359065370000000000007760018008170000000110000012940000124320000160074000008700
SP122Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Peptidiphaga;sp. HMT18300000000000000000000027400000000000000000000000000000000000005150022841007522970007700000223916810014920000000000000051210810063377900763000000000
SP123Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;vestibularis001700000007200000000001040000000115000000000000000000108000000001800000000015000000006000000000053000000000000000000400000000
SP124Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Schaalia;sp. HMT172390434018605202893300138133000000477353392760003771900020300000021981211000003020000196122067400000000000000000000000000000000000110000000000000000000000000
SP125Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidales_[F-2];Bacteroidales_[G-2];sp._Oral_Taxon_27400000120000000001602400420005100000000000000000000000000001301700820000047042014000933200000000000000024690000017040000000030520030120049
SP126Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT3095600000000013250019070000000000000000017000062000000000000013058000000000000000000000000000000003700000000000000000000000000
SP127Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;endodontalis00000000000000000000000000000000000000000001200015000000000000000164606400000016700054000061410500000003300018980019615800107000000000
SP128Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;Cardiobacteriaceae;Cardiobacterium;valvarum000000000000000000000000000000000000000000000000000000000004400007800000043000000000006100520006600000000000000320330081000
SP129Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;histicola368119252194044300706112013254630015700250333109216437261035882890158102000457756588763521256520138146016257148235649198001322442300000000620000000020200000463000000000000550000370000000000000550
SP13Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;paradiacens24722335113449730041919810513512623011183923575862747426773142412761603237104429831651131542733624838531724234165892177369040865136455054465846130734732618435524713433520591970731411531271591628800003713413063164000701178713805094591668802509601400136520021203260142221270120632698900
SP130Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._Oral_Taxon_180000061000000000000000083012000000008000000000000000000000310000000085000106002912220001560000018792808500006181640000124000043610000000380000000250
SP131Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;infantis_clade_4310001021617104900022102186255890178950015341000009518103201590641600001990005503711410528052178180193000000000000000000000000000000000000000000000000000000000
SP132Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;oralis_subsp._tigurinus_clade_0700260037400000902200002700400039900000173100000120000078000005600000058002903515300900141550400000049061328008600179113380740024300370997000120018816340000550
SP133Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;sputorum0025005700000000000000000062000140390000000030000118160000000000000000000000000000000000000000000000000000000292000000000000
SP134Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp._AF189244.10082070000000056000000002060804100000000000000000380000000000000051033100382012604411336415120000017300012736622015579132100012932512528600262181241880140212030886342038186000
SP138Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT2250000000000000000000000000000000000000000000000000000000001150000057329082000004300000000000000000000001200000000087000000000
SP139Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT417110000056000000830440000400020600000090000000000010712800000142900000124720099000000000000720000042000000000000000000000000000001670000000
SP14Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Selenomonas;sp._Oral_Taxon_1370000000000000000000000000000000000000000000000000000000000001670000000000000000001070000000018300180000001350000000071000000
SP140Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Dialister;invisus0000000000000000000005295500000000000000000000000000000000000002700000000027250790910001091311310296290000066115006400330031100098013804400709093
SP141Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Shuttleworthia;satelles000000000000000000000000000000000000000000000000000000000000055000000005808000000000000000000008000009500000000000000
SP142Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT175012600000000000000000013713000710000025312700000000000000006301010000000127277850046063001915965034049022374146075498001517762651371650018703092420100516031910934110637967001211570120
SP143Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Stomatobaculum;longum132000015200000000001790000451160141013391079050000166300019400106000095000000000008300000000990001130000048005400000000000000000000000000009700
SP147Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;sp. HMT2040000000000000000000000004400000000000000000000000004500000000000000000000002200000580000002800001300124079000410051000000000115
SP148Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Fretibacterium;sp. HMT3600024000000000000000000000000000000000000000000000000000000000000000000005800000000000000000000000000000000000720000036
SP149Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;gingivalis700180052000160450507300000001080000000015942540000004800180022000490000012066280046155411210530008874017469490014809706019800540151047641823911075330721602415362003902325200
SP15Bacteria;Actinobacteria;Actinomycetia;Propionibacteriales;Propionibacteriaceae;Arachnia;propionica0450000000000000000000000000000000000000000000000000000000209112000210031502550000178237122033139310000602090000012007927100789086175006770002288818823001321921060943
SP150Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Ottowia;sp. HMT8940000000000000000000000000000000000000000000000000014000000000000044000061002370000000000000000001590000000000000001610083000
SP151Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT2230000000000000000000000000000000000000000000000000000000000000000000000001300000000000000000012300000000000000000000000
SP152Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;israelii00000000000000000000000000000000000000000000000000000000000000000000144770000000000001000034000179000000000103000000133000000
SP153Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;sp. HMT14900000310026000000031000007800000000000000090068000000000028000000000000000000000000000000000000000000000002000000000000000
SP154Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;oulorum000000007500000000000051000008800000000000014000005007000000001000710000003028131830546500030761451420110000102630085000063018000009706046000241500
SP155Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;sp. HMT86400000000000000000000000000000000000000000000000000000000000000001859035004101010000000270000000078012400000019000000042000696900
SP156Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;buccalis00000000000026400000000000000000000000000000000000001030000000910000027401580027800176210000110000001540002890021900000000000009501414300012100
SP157Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;massiliensis000000000000000000000000000000000000000000000000000000000051516038156024582263219510024115213400145138017105311470124373339001021970000003220383924611490287226000041421100
SP158Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum_ss_polymorphum00000000000000000000024000000000000000000000000860000000000770380001743000008490154761701301040000056116002361610016716300779201110010400015801464100693200
SP159Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp. HMT0571471500490001052700440148130555114315030105210000000540179410347114216801030000000311512161291908273000000000000000000000000000000000000000000000000000000000
SP16Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;concisus022010716618012231720364400750111054134402931961435622312002700615010719755003625488162847270176948762364311392004991000007366586600140001331030000000114130126224103219034580006203200007763113240000097017
SP160Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Lachnospiraceae_[G-2];bacterium HMT0962280000006708984000009600980000007800690000000016701320000001739100065019700000000000000000000000000000000000000000000000000000000000
SP161Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT4480000000000000000000001500000000000000000000006500498000000000013680019660000884000165366987402128103000017561021073600258871800104911506039010000554599069500497881306390013410351131469
SP162Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sp. HMT01800000000000000000000000000000000000000000000000000000000000000000000000000000000000000000004220000000000000000000000
SP163Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Micrococcaceae;Rothia;aeria23652000337700029000000085200000604700310246704100044003600027200440582308900000027001423221050069000111174006475843000298371230337508844171954109950149561902539417902349109006741050059
SP164Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Lachnospiraceae_[G-3];bacterium HMT1000000000000000001010000000000000000001100000000000000000000000000000000380000000000000000000480014000000000000000000000000
SP165Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Lachnoanaerobaculum;umeaense000000000000000000000000000000000000000000085000000000000000000021006400210000000520000000041000000000000000001053800000000
SP166Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Schaalia;sp. HMT8774018000000000000000000554000000000000000022100000000016000000102000000005200130050130206002190060000001000080183000000122600000002004700014400
SP168Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT21500000010314200500106668546008700000017534000310172631280061110088001031679300184321817601640005600000240000001090000250000007500000000000082096000011600570018900000
SP169Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;constellatus080000000030090001500039000000000000000002000000001510000001500000000000080000850150000000000063000700000000000000142000027000
SP17Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;naeslundii000000000000000000000198025000000000000000000000000000000000167023504440946346205797461631653360765121004671095773620691685740432264197399880862980021337156811130730470218017120459274131340581
SP170Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT49800000000000000000000000000000000000000000000000000000000000000001040000161007900000266000031500000000000000000000165015700008400
SP171Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;sp. HMT3081211400329202216662501203160003600000517848255635379350082132520011854017003082015771218143868913432730720000000000000000000000000000000000000000000001300000000000
SP172Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-6];bacterium HMT87000000000000765100370000000000000010610400000000000000000000000000000000000073180003400000000000005590730000000000000000740009100
SP173Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;sp. HMT277000000000000000000000000000000000000000000000003800000000000000000048000006200000000000000000000000000000000000000000
SP174Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT317058000000000000000000000000000000000000000000000000000000003500126000000000051881000000001366000530002620000000014700001510281000021500
SP175Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Bergeyella;sp. HMT9000000000000000000000000000000000000000000000000000000000000000000810122000003613000000009200060002611600150000000000012000002000
SP176Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sobrinus000000000000000002500000000000000000000000003800000000000530000000000000150000880000000000000000041810000000000001820000041830
SP177Bacteria;Actinobacteria;Actinobacteria;Bifidobacteriales;Bifidobacteriaceae;Bifidobacterium;dentium0000950000000000000000000000000000000000000000000000000000000019800000000000018600000000000000000011200000000000000000000
SP178Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Catonella;morbi000038310000000004245000003804703800000024135000022104004563018000240960340000000051107000000521130000000002700000006900000391800000006500004320035
SP179Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;endodontalis001530130000000000008500018200000000000057029000810120005800000000840000011001080088012500370000001500000000001898700270000000000084250006800247
SP18Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;parasanguinis_II145445250419259772329449379671160338243540354023513329118184935302575997663522634990275309663391434328364548656923351613855146528057257519227684293699234951886084261500070030165700000712006910001301370036000096000046941600390120921090000120004030
SP180Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;rava000000000055000006600000000000220084000000000000000010100000000000000000000000000000000000000000000000000000000000000000
SP181Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Schaalia;sp. HMT1800000008324003414100566938500000700085000001890000000612270000000721354302613741002700000000000003020117000000007900000000380000000000000000000000
SP182Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella;oralis0000660000000900000000000000010002400000000000000050070000025890005313503105514504800039015020243001491271200079045726005342525820116242023678196813000362759520200
SP183Bacteria;Gracilibacteria_(GN02);Gracilibacteria_(GN02)_[C-1];Gracilibacteria_(GN02)_[O-1];Gracilibacteria_(GN02)_[F-1];Gracilibacteria_(GN02)_[G-1];bacterium HMT87200000000000120000000000000000000000000000000230001100000000006800013053020600000334000011200013400055000000380041000015301501820251220012430055
SP184Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT392021000000000000000000000000000000000000000000000000550000000000000001120000017200000000000030960000000000117000089056001810009200
SP185Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum_subsp._vincentii000000000000000000000370000000000000000000000000000000000000000000000000000000000000000000400000760000000000000062000280
SP187Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Aggregatibacter;segnis000000000000000000000000000000000000000000000000000000000000000000000084000000000000000000001010000000000000000000000
SP188Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae;Abiotrophia;defectiva00000000000461790000000000089000002380000000000000000127088072000000000008500004070163000062000000020400023006060000009200000000036602882430000
SP19Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp. HMT0660112115310020410138450492601916008407017204001822464494474633093130018010401001547022199334561422860134947086023100000052036000001480000000000000000000000001500001691000005200000
SP190Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT47200000000000000000000000000000000000000000000000000000000000000000000000000000000000000232000000032000004800002200000000131
SP191Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Corynebacteriaceae;Corynebacterium;accolens00000000000000000000000000000002760000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP192Bacteria;Saccharibacteria(TM7);TM7_[C];TM7_[O];TM7_[F];TM7_[G];sp._Oral_Taxon_A560000068037000000005100000000015000000000000780000342956000000435200000000000000000000000000000000000000000000000000000000000
SP193Bacteria;Actinobacteria;Actinomycetia;Propionibacteriales;Propionibacteriaceae;Arachnia;rubra0032000950048000000000004400000025000000000000045000059000000000001820820171800858102327014401358800176024000216005601078005610286480028014806140206200182378003607655700
SP194Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT2120000790000000000000000000000000000000000000000000000000000009700028912406607300033000011100001240000006902510000000630000224011200112273000
SP196Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Schaalia;georgiae000000000000000000000270000000000000000000000000000000000059140000191603100003113439000000001280000000351330027007000000000001080351580
SP197Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-2];Saccharibacteria_(TM7)_[G-5];bacterium HMT3560000000000000000017000240000000000000017000000000000038000033000000007500079752014815406390002060000000000019100545004200000000003720030000
SP199Bacteria;Firmicutes;Negativicutes;Veillonellales;Veillonellaceae;Veillonellaceae_[G-1];bacterium HMT15000000000000000000000000000000000000000000000000000000000000001340000000000000000005100000000000000000036000000000039000
SP2Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._Oral_Taxon_1710000000000000000000000000000000000000000003100000000000000000931140000005100000008301001360000000000000270000970220139007502700000
SP20Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;odontolyticus16016407214214575618825290140940551392936299020319812413840117521017182163853243910209136628845042038828615295567733264390434291533225759134242421346399348070300000010801510113013700000038000022700081000000068000000155000000010000000
SP200Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Coriobacteriaceae;Olsenella;sp._Oral_Taxon_8070000000000000000000000000000000000000000000000000000000000000000930000000000000000000000000277002970002800026000000000000
SP201Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;aurantiaca0000000003700000183000000000000000000126000000000000000000113000000000000000000000000000000000000000000000000000000000000
SP202Bacteria;Firmicutes;Clostridia;Eubacteriales;Lachnospiraceae;Lachnoanaerobaculum;orale0000000018100000000000002480970024100007700000000000001600152000000000000000000000000000000000280000000000000000000000000000000
SP203Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Tannerellaceae;Tannerella;forsythia0031000000000000000000000000000000000000030000000000000000000000000000000000000000000000000000000065000000000000024000
SP204Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;sp. HMT4730000000000001820014100900000000000010800013400000000091000003500145000000000000003000000000000000000088000000000000000005800000000
SP205Bacteria;Firmicutes;Negativicutes;Veillonellales;Veillonellaceae;Veillonellaceae_[G-1];bacterium HMT155000000000000000000000000000000000000000000000000000000000000970000000001130002100054141000000000019001500000000000000000000
SP206Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Corynebacteriaceae;Corynebacterium;sp._Oral_Taxon_A160000000009500000000000000000000000000000000000001650000000000000081000249044000000000000111001630500000000002340018600017803000044000
SP207Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcaceae_[G-2];bacterium HMT08572000110000000023220360000000000000001822000010401020004000310004625620000000000000000000000000000000000014000000000000000140000000
SP209Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Eikenella;corrodens000000000000000000000000000000000000000000000000000000000000000150115000570006500000000896700000760617800058360002424500414101470069000
SP21Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;koreensis00000011000341000160128000000000034002220009199000513900000560036600024830002200000000024000000000000000000140000000000000000750340003400000
SP210Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT89600000000000000000000058000000000000000000000000000000000000007000000162000980105000000162000919004500010600000000000000000000000
SP211Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-1];[Eubacterium]_sulci00000011600420000011111702700052001054700290025049000450280000240049000000066000000000000000000000000000000000000000000000000000000000
SP212Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT8970000000000000000000000000000000000000000000000000000000000122000002670000000000000000000001210000000001100000000020300000000
SP213Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp. HMT0741213134007796133385319108156013118104005170001417766049158108180109110001000009728816691310410628905231200148000000019000040000000000000000130000000000000000000053001000
SP214Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sp._Oral_Taxon_1400000000000000000000000000000000000000000000000000000000000146000000145000000000099000930000000000000008800157000001180000000
SP215Bacteria;Firmicutes;Clostridia;Eubacteriales;Lachnospiraceae;Lachnoanaerobaculum;gingivalis0000770000000000000000000000000000000000000000000000650000000000000000000000000000000000000000000000000068000006400000
SP216Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Mogibacterium;neglectum000000000000000000000000000000000000000000000000000000000021068830000003400274400000019113058000010205800000078009200000000000000
SP217Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT5250000000000000000000000000000000000000000000000000000000000000000008600045104460000003030000000000106000003950000000000000000753
SP219Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum_ss_animalis00000000000000000000000000000000000000000000000000000000000000000000000000370000014800000000000051000001300162000000001090800
SP22Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;Leptotrichia;hofstadii004800000000000000003094001400000000000000000000000000000000230850049142001716904315064000053870116029188150006100020400000034018500025001651333700250000
SP220Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Selenomonas;sp._Oral_Taxon_E83000000000000000000000000000000000000000000000000000000000000000038000000074680000009054300620000320000000170000020000000000
SP222Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;sp. HMT91400000000000000034000000000242000000000000006100046000000000000000000000000000000000000000000000000000000000000000000000
SP228Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;parahaemolyticus0000035490000300000003800700319000000000000000000042000033022000000000000000000000000000000000000000000000000080000000000000
SP229Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;sp. HMT137000001000000000000000000000000000000000000000000000000000000000033000002700250000000003039000000081000000000000000120020000
SP23Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;cristatus7915898222421313001031471752354051234115580084117681062592114890591112622592161023004765293392901250491123118205250420611709225365301561001000584629159390262425465328100399597157152051615188252067385617612219226488182363524413428200774191241360175306184259394052
SP231Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Lactobacillus;ultunensis000000000000000008400000000000000000010000000000000000000800000000000000000003400000000000007900000000000000000000000900
SP233Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;sp. HMT03600000000000000019000000080000350000000000000000001210000000000000000001500000000000000000000000000000000000000000000000
SP234Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Schaalia;meyeri000000000000000000000000000000000000000000000000000000000000000005900000000000000001050000008900000000000001080000000000
SP235Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Pseudoleptotrichia;goodfellowii000000000000000000000000000000000000000000000000000000000000000000520000032000000000000000000420036000000000236600000000
SP237Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Megasphaera;micronuciformis16326097100482511703628056670223000363825100011168063276494750139840138541330000880014000134119412060870000000000000004700000620000000000000000000000001600000025000
SP238Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;saccharolytica000000000000000000000000000000000000000000000000000000000000000000400000010200000000024000490006700000000000004866022000000
SP239Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Tannerella;sp. HMT8080000000000000000000000000000000000000000000000000000000000000000000000141170000000000000000000000001600000000000000000
SP24Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT1700000193000000010100000000670000000001010000000000770000000950000500085047599300135001250854900002000002383305000882887480640001180318384433332527305700730152373900021
SP240Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Eubacterium_[XI][G-7];yurii00000000000000000000000000000000000000000000000000000000000000000000000076000000000000000004100000280000000019000000086
SP242Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;flueggei000000000000000000000000000000000000000000000000000000000000694600000000610000000036800000000000000000214000000000000000
SP245Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-6];[Eubacterium]_nodatum004400000000000000000067000000000000001500033000000000000000000000000000260000000000000000000000000000000000000000000180
SP246Bacteria;Absconditabacteria_(SR1);Absconditabacteria_(SR1)_[C-1];Absconditabacteria_(SR1)_[O-1];Absconditabacteria_(SR1)_[F-1];Absconditabacteria_(SR1)_[G-1];bacterium HMT8750000006900016280000280760000001370000170000000000000021028108000038000000000000000000000000000000000000000000000000000000000000
SP247Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT30000000000000000000000000000000000000000000000000000000000000011000000000014937310960001295600415000003480005300000000000000000000
SP248Bacteria;Firmicutes;Negativicutes;Veillonellales;Veillonellaceae;Anaeroglobus;geminatus000000000000000000000000000000000000000000000000000000000000000000000000006800004400000000000009200000000000000000000
SP249Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-1];[Eubacterium]_infirmum000000000000000000000000000000000000000000000000000000000000181280000004002304900000024001800000001300000000200000031087000000
SP25Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XIII];Parvimonas;sp._Oral_Taxon_110000021000000000000000000670000000000000000001400000000000000100000000000009300370000007000000000000003100002050000000034440570127
SP250Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT21820600000035000000009700121000000990000000000014001600000570000000023000000000000000000000000000000000000000000000000000000000000
SP251Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;gingivalis0010200000000000000000019500000000034000000001180000000000000000000000000000000000000000000000000000000000000000000000000
SP252Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;trevisanii000000000000000000000000000000000000000000000000000000000000000000000000000000000063000000490000002200000000000000160156
SP253Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;sp. HMT2840000000000000000000000000000000000000000000000000000000000000001850003700000000000000380000000000000000000004460000000077
SP256Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;veroralis0000000000002500000000000000000000000000000000000000000000000000000058032144029000000000220000000000000790000000016000000035
SP257Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;loescheii0000000000000000000000000000000000000000000000000000000000000000001150817400000000000000061210058000000000000000330233000000
SP259Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT41400000000000000000000013900000000000000000000000000000000000042000000011900000021000000000000147002540161000000000000066000000000
SP26Bacteria;Firmicutes;Tissierellia;Tissierellales;Peptoniphilaceae;Parvimonas;micra0321339000018000106340009200321629033003000280002204700072000100140000700300168300720663500113230004740028401123600049153006200130481940198207088910018203200005302620168056673632431
SP263Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;oralis_subsp._tigurinus_clade_0710000000000000000000000000000000000000000000000000000000000000000133000000000000000000310000000000000000002500000000000
SP264Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-2];bacterium HMT35000000000000000000000000000000000000000000000000000000000000000008800000009400000000000000000000000000000000000000000
SP265Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Schaalia;lingnae_[Not_Validly_Published]00000000131000000002370000000000000000000000000000000000000280000000000000000000000000000000000000000000000000000000000
SP267Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIVa];Oribacterium;sp._Oral_Taxon_78000000000000000000000340000000000000000000000000000000000000007400000000000000004954800000000000250001300240000008300000026
SP268Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;Leptotrichia;goodfellowii000000000000000000000000000000000000000000000000000000000000000290000000002500000210000003800000000000000770000000007400
SP27Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Atopobiaceae;Lancefieldella;rimae0000000000000000000000000000000000000000000000000000000000004569500000000042730000001800022000000000000060002260000000000501100
SP271Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;sp. HMT1360000033000000000010700420091000490000000000016100700000000000000000001150000000000015000000100180000000000000000000000000000000
SP273Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;maculosa000000000000000000000000000000000000000000000030000000000000000000000490010000009100034000000062000000000000002828000015028
SP274Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Butyrivibrio;sp. HMT45500000000000000001120000000000000000000003500000000000000012700000000000000000000000000000000000000000000000000000000000
SP279Bacteria;Absconditabacteria_(SR1);Absconditabacteria_(SR1)_[C-1];Absconditabacteria_(SR1)_[O-1];Absconditabacteria_(SR1)_[F-1];Absconditabacteria_(SR1)_[G-1];bacterium HMT874000000000000440069000000000000000000000000000000000000000000011000208504700280045000000000295400710001010100000210040006600000662200
SP28Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-1];bacterium HMT869000031000000000000000000000000000000000000000000000000000000001280000000000249820000000000000000012480000000000000000001249400
SP281Bacteria;Absconditabacteria_(SR1);Absconditabacteria_(SR1)_[C-1];Absconditabacteria_(SR1)_[O-1];Absconditabacteria_(SR1)_[F-1];Absconditabacteria_(SR1)_[G-1];bacterium HMT34500000075000000000000000000280000000000000000000009944000039000000000000000024067000000000000000000000000000000000000000018
SP282Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;sp. HMT336000000000000000000000000000000000000000000000010000000000040000000462801190003700001150000153064003900423000000000011557099250750000000
SP283Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;elegans00000000000194650000000000000000025200000000000000002403100000000000000000000000000000000000006200000000000000000000000000
SP284Bacteria;Firmicutes;Negativicutes;Veillonellales;Veillonellaceae;Colibacter;massiliensis0000000000000000000000000000000000000000000000000000000000000104001400000000000000000000000000000000007800000000000000
SP287Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT304007500000000000000000096000000000000000000350000000000000000000000000000000000000000000000000000000000000000000000000
SP288Bacteria;Bacteroidetes;Bacteroides;Bacteroidales;Porphyromonadaceae;Porphyromonas;sp._Oral_Taxon_B430000000000004000000000000000000200000000000000000000000000000000000008637130000000000000000710100000000000000000000000000
SP29Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;melaninogenica69718615561263111341565879148836530214393901886406237480520015507407481708900367105822820613651223484789587429087138324210003447311267853187913362326329371571256935423520152202100005900760162139150027650000042416000011002100850000000079102001000001990003120093
SP290Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;shahii00000000000000018700000000011000000000144000000000000230000017000000000000000000000000000000000000000000000000000000000000
SP293Bacteria;Firmicutes;Clostridia;Eubacteriales;Peptostreptococcaceae;Filifactor;alocis00610000000000000036000610000000000000012000450000000000000000000000000007200000130000000000000000000000000000000000037000
SP295Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;pleuritidis0018000000000000003400000000000000000000000000000000000000000000001300076000000000000000000000000000440001300009500008100165
SP3Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT22128800810360054307350740086080006790020100036646000004600003876604220000192500511230859029600000000000000000000000000001260000000000048000000000000000000
SP30Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT3061479900000000529200001980008100000116001500000056400121690000059002150000030400000000000000000000000000000000000000000000000000000000000
SP301Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Mogibacterium;timidum0000040000000000014000000000000000000000000000000000000028000007600000078000000000138000000000000014000000000000000000200
SP303Bacteria;Actinobacteria;Actinomycetia;Propionibacteriales;Propionibacteriaceae;Propionibacterium;acidifaciens000000000000000000000003900000000000000000000000000000000000000000000000000000001890000000000000000000000000000000000
SP305Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;sp. HMT33500000000000000000000000000000000000000000000000000000000000000000052000000002900000000000000000010600000000077000000000
SP306Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;shahii00000000000000000000000000000000000000000000000000000000000000000000000000000000000000019500000000000000000000000000
SP307Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;infelix00000000000000000000000000000000000000000000000000000000000000005700000000000000000011000000120000000000000000000000091
SP309Bacteria;Firmicutes;Clostridia;Eubacteriales;Peptostreptococcaceae;Peptoanaerobacter;[Eubacterium] yurii00000000000000000000000000000000000000000000000000000000002500000530590000000000000000000000000000000000000002200002400
SP31Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;parainfluenzae16440980404779241080511319106843742831744991649023409910387694097480296152423111275617719306223575510076391935751278494479481026128010704872041110635330118060100976001356681890141050178000310401000073370124114010143024710306600122001230891786471115020441310651657753000
SP32Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;oralis0180288342298227226629722251211839346231232101919402321751313694051310649118815743421151570165010110580711171883291462718268200205928461536437025314612143265299196205454098624711652800121435326982681501879884153601243513093154714118014016128539036070468143217012324814613507273991
SP328Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIVa];Lachnospiraceae_[G];sp._Oral_Taxon_B320000000000000000000000000000000000000000000000000000000000000000750000000000000000000000000000000000000000280270007100
SP33Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;oris000010656140000075000059000510252000000000000000000122000490004300005600002245440460000117104136150872247000014313727304710220016101121502140960000042618300710183011905817616100
SP334Bacteria;Actinobacteria;Coriobacteriia;Eggerthellales;Eggerthellaceae;Cryptobacterium;curtum0000000000000000000000000000000000000000000000000000000000000000000000001030127000000000000000000000000000000000000000
SP339Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella;denitrificans00000000000000000000000000000000000000000000000000000000000000700000000000000000000000000000000000002160000000000000
SP34Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;gracilis00000000000000000000035035000000000000000000000030000000000000014111002500005836966198014472877196138003800730007410764203504922112004301232061510020680088
SP342Bacteria;Firmicutes;Negativicutes;Veillonellales;Veillonellaceae;Veillonella;sp. HMT917000000000000000000000000000000000001580000000000000000007900000000000000000000000000000000000000000000000000000000000
SP35Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;noxia0000003700000000000000710000000000000000000000006900000000006203215300150780033026526615091670011008520220972159700228006242410500012501650087001650117082000000
SP36Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sicca00000000000000007304990000011300000167000000000000000000000002800000001190005202807950830000870000009703940610172017000005700000005096400000000000
SP37Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum002520000000000000081000293000002900000000000000000019900000000139001621201121690254001330898415011025445400010201260108406490018726001161883172390003191420012900645230029619301722446910680
SP38Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;infantis25923126310722957763557221464531321182167155642367023652443206084212447301116303565222257147641216704362869053547107129237633143404316185356144167164119035200000045147045052000130023000000000000000000280000220145412000008100000
SP39Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Schaalia;odontolytica11016100015311858018532611011563620803021001590592003472301421860056450184000285168834515810165312310021974601321037663072000000012600067000000000000002320001300000001090000000301000026000000
SP4Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;adiacens000000000490000000000000187000000000000000000000001140000000009501541404215615025728310318219086143781238202551140008719923600126023522524513815927200001619901511413198901850116181225322203095
SP40Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;pasteri0901303969019311503361652439723003742560119092780146024932026012630809901651902200292533801203743540148196541852402599401190014071008200205861420240016300000021661690014530513700028713724000246000019309701102890015300
SP41Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;nanceiensis0281100083001790005567916708500000228600145270061412300340308905648399108228043270000000000000000000000000000000000000000000000000021000003700000
SP42Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;denticola00002401000000000005400020105700042000000000000000000360006000009500001981600000052132201017503710025141612982058000007300084449040000780003320008000139000
SP43Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Bergeyella;sp. HMT3222425490003600620182904001203801300273200025120000170055230860006300350000130000247014725082961230553285001384600640980008301030107410004692290828003632078195460134000015817323024
SP44Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Lactobacillus;salivarius0000000000000000030900000000000000000000000000000000000008180000000008500000000241000000000000000000000000000000000000000
SP45Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;bacilliformis000000000090000000000000000000000000000001400000070000000000000556000096000000000000018100001120000000977001330417000000006000012
SP46Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;catoniae00000000000011300000002600000000000000000000000000330000000000420000134002420011100121000070000081001170012092100001150000023000017304473058197174075
SP47Bacteria;Firmicutes;Clostridia;Eubacteriales;Peptostreptococcaceae;Peptostreptococcus;stomatis0000807419000250020770025000003062000190020154900000690164540430053000022000042008300000069000154000000000000000031000000125000000000000034000
SP48Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sinensis013000000000034000000030000000000000000000000000000017000000016300000000005000000000000000000000000000000000000062000000
SP49Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sanguinis65178206068713910047190137125161317508303003394310702841221201142277301071510217000605116004771453425117435166651425601404532428390755198136366926773895341028605793106492212130714932475700142204093556169235017333821710833288040047124421009169178426112896288881782441288396062133624981780921
SP5Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._str._C30016916761600279140203341123442934702190059794272162919100215104512841126219512400052329027468212710015618410816112221732623563703887923911884943256249072026085058366211712568931723651642761039727933321504477004921403099201304268447123257839917616192799706331803146210028562513563243211022590349118591178129163343
SP50Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Micrococcaceae;Rothia;mucilaginosa124953342002614318614281730116720828021119587153514781031304624216722322699412459200539321001885765141763399519088651028147591725621609792712537032423056129120739882643639209619333028859162431095114000000001670108000000022400000000000000000000000016320404350000012100000
SP51Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium;periodonticum210201025101566912040136169163196272293694980687012000022953824001362351220543285733002850794953221323246803880167429020126031769610102020000000000000000000000000000000000000000000001550000014400000
SP52Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;sp. HMT38000000000000000000000000550000000000000000000000380000000000000190000000000000000077000000069000000000011322000041830000002500
SP53Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;mucosa000000000432303230000011800000000099002090000033000000650013000000000000000000053532000300000243000000000156600026200210509000000575000000430002300
SP54Bacteria;Actinobacteria;Actinomycetia;Micrococcales;Micrococcaceae;Rothia;dentocariosa21928226011877669030754109099087054542165290315011852443002813589682690104001031115901830279812060209252214613204200157958804890137618630711162401893600122026831853313118206520433896376317900497034424637136406169883232982960589308821352552092760972071418100
SP55Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;Cardiobacteriaceae;Cardiobacterium;hominis00130000000000000000000009000000000000000000000021000000000000001931014002021430120013240006670000127055080103042100024000596601030019610900258166100251900060
SP56Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Tannerella;sp. HMT2860002200200000080163700007000000000000042000000860000001302000390000003900352305424075118099610000033002883009810200502200000842600034043981331500027082
SP57Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;salivae20700058147001840120102100000178000060132101127481082164021301093400119001140218431529909726539240640116044258014200000000000022200000000001430000007200000000000000000000000000
SP58Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-1];bacterium HMT3460000000000000240000000770000000000000000000000000000000000001105873408202540164003652227841682720000073012000545005651350872090047048029100222000003701822520008616222
SP59Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Oribacterium;sinus12385482411691541726673670895217115766215072011701711241428912000875008717230017071471141362662091715061270011570560284016400000000194016000000000000000016629000001100000000000044000000001900
SP6Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;viscosus00158000000000180000000007100000155000540067003500830000048036000000072008100003004650020511513614970741510032000001006000029107280150403000003922415369147000654611400507
SP60Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;Lautropia;mirabilis03500134164600000062000000000039316801100005112500000000007107800160000000000090262092140177677074992035009401063508008600011153255303260010492603940087137531480137615752250764027601181147128199030
SP63Bacteria;Actinobacteria;Actinobacteria;Bifidobacteriales;Bifidobacteriaceae;Parascardovia;denticolens000000000000000000000000600000000000010200001600000000000000000000000000000000245000000000000000000360000000000000000003710
SP64Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;dentalis000000000000000000000129000000000000000000000000000000000000450207803110002820005230482560000015909910021000859028402050000274000013500048100000000144
SP65Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;sputigena00000000000000000000095300000000000000000000290000000000038000005109400850006885700689879000331228333011718000002889000006306085000008500004820100
SP66Bacteria;Actinobacteria;Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium;durum063000000052000000000000008400001190000000000000036009412100000000000009303190111299000095000000004030132852360000036600008846001710830951380000261000
SP67Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;leadbetteri02439033000000000068009200110000140000000280000012120000010300830011700000710048206113068049000122006106300007520909214600077620035005300680206079477217002889120060
SP68Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;elongata026000065001900169000000000000000110054000000000000190051001020000000810000964337021746450601374600083000652068690258870107302040000000039158407067200690196199000
SP69Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;wadei0000176120002007563000119560057050610000133000000125000000103124000008800008200011700117122312120000006963470040500259284390766501179044100270312001990144720243018738917524201040133233018300038100
SP7Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;jejuni06900000007810295001310219000370211000400351104800100039002043361252000400782240000036600000000000000000000000000000000000000000000000000000000000
SP70Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Solobacterium;moorei4900000885000445736440109177000037123827950560942001206152000840791027783638634859337187557949000000013300000000011160000001272525012000013800108000005600158000001150650000018
SP71Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-3];bacterium HMT351082600389200000003002060001707012990000000000000530180004357490041000396839015000391080290005600760015800000370014100003502020500000143200000000000008800
SP72Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Aggregatibacter;aphrophilus00000000000000000000000000000000000000000000000000000000000000000000108000000000000000000000000000000000000000001382300
SP73Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-1];bacterium HMT9570220000000491200000000190000000000000000000000000001200000000019012500000330104000232000085044000000023604100000000005700005600000000
SP74Bacteria;Firmicutes;Bacilli;Bacillales;Gemellaceae;Gemella;morbillorum02231000140023070670041000003700000017067000000000290000031284402900000032000002101120315616201704230924100000370019014700038009515725036036083850081156700182983370035730300
SP75Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;flavescens|subflava3900850504077013761090222422525111911094250182600000896000121766922213709434903220012300525324081995580421038105684807428847534000000000000373000000000000000000362000001600000000093077024600011900000
SP76Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;salivarius301361413149349451942569111951871087175660543154267320778212869820835912685937844453941752140667183731140712873323818252127089201254683718562615739723948250920140876726831670232481421606592566313333884659192048130675500001084100000130332002002600011102500323000216670010101174205017040000000077512
SP77Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;australis007227700140526247035311935216425459225065500000672908102061481360542278187162459041267903223014358411926417420302161570460000000114010000000220750000000000000580000000000286319103300054700000
SP78Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._Oral_Taxon_B66102000000150008000000083000024023000150000000017000000000000000007341200000117800070000000059823000820337000120000000355861000006500000000000
SP79Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-1];bacterium HMT3490000000000000000000009000000000000000000000000000000000000018900000147000182370740516000012900002500370007400002073000001000165022300062200
SP8Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;graevenitzii37700269032503684830907619850712745851900340612003730002431480351750528181263141001925519000003041758119010345293216832028400000000000000000000000000000000000000000000000000000000000
SP80Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT1690000000000000000000000000000000000000390000000000000000000000000000072040000132000220000011000000006915801268117000000040000000322
SP81Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcaceae_[G-1];bacterium HMT07502900000000005600017600035000001000000006900000000000036610000715000016000005900800000951790000000040760001190020200000000000000000004395020
SP82Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp. HMT056014000000000000000000000000000000000000000009000120000000000000006314106300003800000000003337600710000000000000560250005800000015
SP83Bacteria;Firmicutes;Bacilli;Bacillales;Gemellaceae;Gemella;sanguinis15707511250954761036321416777941184724272622121633874850033158959713306309010017963116563568931242661810254466892316848025012024603541961230330000000169301020000000805200000240200000014000017700000277278000008900000
SP84Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;tannerae0350025340000007900780000451010266600002601500350000019000000065090028000001820023701050000166001681110000065000580009500051400000273000000690312012400169037186
SP85Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;anginosus0126000000000250400000163561090000017260000000030016000330201616060012800901233240003370024677000581010405102819300000363750016002813950051030428070243033769900094260
SP86Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._str._ChDCB1970000259000001560000009600015103231370000101000000153080000630001720129000000042165460027068104213471121360001670001012470694014740203155028012713302901307805826240201351568176393230416151169000026100503
SP87Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;mitis176438552265815547743420941890146084913211111497469662674654061688741342190822164673674646020667646733631943350841730770031655257357609727835833649050663334712803421829018178193701943291500294006681611790007082542055038266149002053670288284771900127986211676194700112
SP88Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;mutans453021700000100000000046143410000040050000017004008000000000000000049200178350001710003350000008627559452330020500000007550115560436171220018500600000000
SP89Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._Oral_Taxon_17800000180000000000000000000000000000000000000000000000000002151110005460154000090974621335000000019610527500000000000000000013400001680002000
SP9Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;chosunense0175600210380650180028610000000183240254000201300211300000000566703900800185011610000012046000107000134000000025009700388300071000000000090121006802601704883100000
SP90Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;intermedia008700000000010700001590001830000000000000064000670000000001300000107000000001670000900000000089000000000000004200000000000001610000640
SP91Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella;atypica44935914869510540214339468002602358828321404403540139401322133615921115552078304151020252327010373451615723174913322331821563024956845227419344723406443294550000000000006200001580000228000000000000015800009900000000000003450
SP92Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;ochracea0000000000000000000000000000000000000000000000000000000006500146000031000543345000018300117000015592200374400008200910012505957000000000
SP93Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;showae000000000000380095000000000000000000680000000000000000000000063540002101600461800020122000000005798151000000000000011200620890905102400553900
SP94Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;johnsonii00001000000000000000044380190000002000000000000000000000000000740113020077316012323751650013276007370001180852000124103000810000040110014753100001571366800141186000
SP96Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;oris000000000000000000000000000000000000005800000000000890000002065227100017730925006400019904002403590340220625407002390338757700583802123373017382020000086
SP97Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;gordonii02418625175026012626568067000000003042814711001879019900160000015000391500155060040028053301446029343774173579130276300127230138006232294815312016504265723316272002730167700157714890042362360285138461502151427000
SP98Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;pallens4765380076890015206064349702300002906680580900104194012614062000018333338468815701710412504712811585000550000000000000000000000000000000000000000000000007100000000
SP99Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT31400000000001121015000000000000007000120200000000000001000060000000000000054000000000000000000000000008700000000000000000
SPN1Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;massiliensis_nov_97.431%00000000000000000000068000000000000000000000000000000000001800000000000017000000010000000000000042832300000000000000000000
SPN10Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;histicola_nov_97.868%0000000520000000000000000003300000000000000000012800000000000000000000000000000000000000000000000000000000000000000000
SPN100Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;australis_nov_97.561%8500076474911431613950001390057038000337726608510133000103006505700054601391212059113019012001000000001702200000000000000000000000000000000000394500000000000
SPN105Bacteria;Firmicutes;Tissierellia;Tissierellales;Peptoniphilaceae;Parvimonas;micra_nov_95.922%00013008200170210006800390000000000000540000000000043001880000039000000000000000000000000000000000000000000000000000000000000
SPN111Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;viscosus_nov_96.975%0000000051000000000000000000570000000000000000006900000000000100000000440770002900300071000000670000000000033000000000000300000
SPN117Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._Oral_Taxon_180_nov_97.099%0000000000000000000000000000000000000000000000000000000004500160055000000012100000000000000000000000000000000000000009100
SPN12Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;naeslundii_nov_97.601%00000000000000000000000000000000000000000000000000000000000320000000000000000052000000001800014000000000000009200000000
SPN129Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._Oral_Taxon_180_nov_97.121%0000000000000000000000000000000000000000000000000021600023700000000000000000000000000000000000000000000000000000000000
SPN13Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sanguinis_nov_97.378%0000000000000000000001400000000000000000000000000000000000000000000000020000006200000000016320180000000000000000004100000
SPN14Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Aggregatibacter;sp. HMT458 nov_97.619%0000000000000000000003300000000000000000068000000000000000000000000000000000000000000000000000000008600000000000015000
SPN141Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;naeslundii_nov_97.601%000000000000000000000000000000000000000000000000000000000000000112000000002700000000000000000000000000008000000000190000
SPN15Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;israelii_nov_94.872%0000000000000000000000000000000000000000000000000000000002650000000000362600001590000000009600450000013900660000000002470480009500
SPN151Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._Oral_Taxon_848_nov_96.571%0000000000000000000000000000000000000000000000000000000000017500000000000000000000000000000000000000000000002110000000
SPN16Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT215 nov_97.490%0000000000000000000000000000300000000003800000000000000000000000000000000000000000000000000000000610000000000000730000
SPN163Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;gerencseriae_nov_97.556%0000000000000000000000000000000000000000000000000000000003300420000007748009731000087005000004000363000007000004208000600
SPN17Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;noxia_nov_97.253%0000000000000000000000000000000000000000000000000000000000000000000000000290000760220000000000000000570000000000000000
SPN175Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Weeksellaceae;Bergeyella;zoohelcum_nov_92.673%0000000000000000000000000000000000000000000000009025000000000000000000350010200000000000000000000000000000000001560042000
SPN18Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT897 nov_97.656%00000000000000000000000000000000000000000000000000000000000000001820000000000000000000000000000000000000000000000000
SPN19Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella;oralis_nov_97.338%000000000000000000000000000000000000000000000000000000000000000000000000000680000000000000000008400000000000000000021
SPN20Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;sp. HMT136 nov_97.917%000000000000000000000000000000000000000000000000000000000000000000000000000000001370000000000000000003400000000000000
SPN21Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;gingivalis_nov_95.914%00000000000000000000000000000000000000000000000000000000000000001050000000000000000120000000000000000000000000120034000
SPN23Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;noxia_nov_97.610%000000000000000000000000000000000000000000000000000000000000000000760000000000000000004100000000000000000043000000000
SPN24Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sp._Oral_Taxon_14_nov_96.970%000002000000000001500000004000000300000000029001200070800002000000000000000000000000000000000000000000000000000000000000
SPN25Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;catoniae_nov_97.710%000000000000000000000000000000000000000000000000000000000000000000000005400000000000000000038000000000000000000610000
SPN27Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae;Abiotrophia;defectiva_nov_96.296%010000162000440006112080001200000003800023410000035000800000037000280001500014040000010802701700038000870001800008307036000008405205600350004500
SPN3Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;israelii_nov_96.507%0000000000000000000000000000000000000000000000000000000000003500001810000000000043700000000000001290000000960000001750158000000
SPN38Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;viscosus_nov_95.849%000000000000000000000000000000000000000000000000000000000081008300000000610141000000019200000000550062000880013008002600000069000
SPN4Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._Oral_Taxon_171_nov_97.778%0000000000000000000000000000000000000000000000000000000000000000000000000000000000410000520000024000000000000000000000
SPN43Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;sp. HMT914 nov_97.250%000000000920000195214000000001600067000041000000000000098000000000000000000000000000000000000000000000000000000000000000000
SPN5Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT300 nov_97.638%00000000000000000000000000000000000000000000000000000000000012900000000000006900008500000000000004300000000000000000000
SPN50Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;israelii_nov_97.393%0000000000000000000000000000000000000000000000000000000000000289002660000000000019500000000000000147000000000000011300000000
SPN6Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT215 nov_97.083%000000000000000195000000000000000000970000000000000000000000000000000000000000000000000000000000000000000000000000000
SPN62Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT175 nov_97.830%0000330000000000000000002000000051000000000000000000000000000103004409200007859000000000099000000104000000000000000000720000000
SPN7Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;viscosus_nov_97.952%0000000000000000000000000000000000000000000000000000000000000000000127000000000000000000000000000000000000001090000000
SPN74Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;gingivalis_nov_97.471%000000000000002600000000000000000000000000000000000000000000000013700830000303700000000800009400000650000063005000078000000000
SPN8Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;parasanguinis_II_nov_97.740%029000000000470000000035000000000000000000200000000001040000000000000000000000000000000000000000000000000000000000000000
SPN86Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;veroralis_nov_97.909%0000000000009312000140000000000002936000012000000000000019000000000000000005510970002600000000000010969000067000000000000000000270
SPN9Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT305 nov_94.071%000000370000000055340000000047000000000000000000000000000250000000000000000000000000000000000000000000002600000000000000
SPP16Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;multispecies_spp16_2000000100000004700051000000100010002800094000000200000004900036000000000000000000000000000000000000000000002000000000000
SPP18Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;multispecies_spp18_200008058002131500001190047000004390007913000010500540000005900173180000260060000000008000000000000000000000000000000000000000000000000
SPP19Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium;multispecies_spp19_2000000000000000000000000000000000000000000000000000000000000000000000017200000001160009700000034110000000000000006600000000
SPP2Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;multispecies_spp2_2000002433480131400070000000000000600004000000000000018180000000000000160000000000000000001100080000000000000000000000000000
SPP21Bacteria;Firmicutes;Clostridia;multiorder;multifamily;Lachnoanaerobaculum;saburreum0000000000000000000000160000000000000000000000000000000000000000650000010510300000240092012441000400001050000000000002402400000000
SPP24Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Staphylococcus;multispecies_spp24_2000000000000000000000000000088000000000000000000000003700000000000000000000000000000000000000000000000000000000000000
SPP25Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;multispecies_spp25_200108000000000000010000210030003000000000001380000000000000000000000000000000000000001540000060000001000270000004400000
SPP26Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;multispecies_spp26_20000000000000000000000000000000000000000000000000000000000000046000000000000000000370000540000000000002200000000000000
SPP31Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;multispecies_spp31_2000000000000000000000000000000000000000000000000000000000000034000000700000000000000000000000000000000000008800000000
SPP35Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;multispecies_spp35_3010330200000069000000000066277000000360190000097005964000000390000024000000000000000000000000000000000000000000000000000000000000
SPP36Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;multispecies_spp36_202699054440652302092240000044278005233870000020726400000025302668000040001580000000550000000000148000000250000038000000630000031000003400000000000000
SPP4Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;multispecies_spp4_20019000000000294915300002390000000003690091043000011000000000030100077000001520000000000000000000000000000000000000000000000000000000
SPP6Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;multifamily;multigenus;multispecies_spp6_296995314809196122105015992013257601271930221501351741111017719689631471381921687380209123621335717710208665125153022284411848391111108239000000000040663531500402280000021500001100258700002030000000009500030000032546
SPP7Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;multispecies_spp7_2250218017610148235321255661672241870192101289203102025020511383122265716068177961509614722578108170891131721281251441982131132515187680093970000551433425510423519018706895202001050001090015202250295701241106501413010530076122157066002141321531672900
SPP9Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Oribacterium;multispecies_spp9_20003700552224417900095753605900040007838091000450790006308773030001147122004950633500000000000000000000000000000000000000000000000000000000000
 
 
Download OTU Tables at Different Taxonomy Levels
PhylumCount*: Relative**: CLR***:
ClassCount*: Relative**: CLR***:
OrderCount*: Relative**: CLR***:
FamilyCount*: Relative**: CLR***:
GenusCount*: Relative**: CLR***:
SpeciesCount*: Relative**: CLR***:
* Read count
** Relative abundance (count/total sample count)
*** Centered log ratio transformed abundance
;
 
The species listed in the table has full taxonomy and a dynamically assigned species ID specific to this report. When some reads match with the reference sequences of more than one species equally (i.e., same percent identiy and alignmnet coverage), they can't be assigned to a particular species. Instead, they are assigned to multiple species with the species notaton "s__multispecies_spp2_2". In this notation, spp2 is the dynamic ID assigned to these reads that hit multiple sequences and the "_2" at the end of the notation means there are two species in the spp2.

You can look up which species are included in the multi-species assignment, in this table below:
 
 
 
 
Another type of notation is "s__multispecies_sppn2_2", in which the "n" in the sppn2 means it's a potential novel species because all the reads in this species have < 98% idenity to any of the reference sequences. They were grouped together based on de novo OTU clustering at 98% identity cutoff. And then a representative sequence was chosed to BLASTN search against the reference database to find the closest match (but will still be < 98%). This representative sequence also matched equally to more than one species, hence the "spp" was given in the label.
 
 

Taxonomy Bar Plots for All Samples

 
 

Taxonomy Bar Plots for Individual Comparison Groups

 
 
Comparison No.Comparison NameFamiliesGeneraSpecies
Comparison 1Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3PDFSVGPDFSVGPDFSVG
Comparison 2Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3PDFSVGPDFSVGPDFSVG
 
 

VIII. Analysis - Alpha Diversity

 

In ecology, alpha diversity (α-diversity) is the mean species diversity in sites or habitats at a local scale. The term was introduced by R. H. Whittaker[1][2] together with the terms beta diversity (β-diversity) and gamma diversity (γ-diversity). Whittaker's idea was that the total species diversity in a landscape (gamma diversity) is determined by two different things, the mean species diversity in sites or habitats at a more local scale (alpha diversity) and the differentiation among those habitats (beta diversity).


References:
Whittaker, R. H. (1960) Vegetation of the Siskiyou Mountains, Oregon and California. Ecological Monographs, 30, 279–338. doi:10.2307/1943563
Whittaker, R. H. (1972). Evolution and Measurement of Species Diversity. Taxon, 21, 213-251. doi:10.2307/1218190

 

Alpha Diversity Analysis by Rarefaction

Diversity measures are affected by the sampling depth. Rarefaction is a technique to assess species richness from the results of sampling. Rarefaction allows the calculation of species richness for a given number of individual samples, based on the construction of so-called rarefaction curves. This curve is a plot of the number of species as a function of the number of samples. Rarefaction curves generally grow rapidly at first, as the most common species are found, but the curves plateau as only the rarest species remain to be sampled.


References:
Willis AD. Rarefaction, Alpha Diversity, and Statistics. Front Microbiol. 2019 Oct 23;10:2407. doi: 10.3389/fmicb.2019.02407. PMID: 31708888; PMCID: PMC6819366.

 
 
 

Boxplot of Alpha-diversity Indices

The two main factors taken into account when measuring diversity are richness and evenness. Richness is a measure of the number of different kinds of organisms present in a particular area. Evenness compares the similarity of the population size of each of the species present. There are many different ways to measure the richness and evenness. These measurements are called "estimators" or "indices". Below is a diversity of 3 commonly used indices showing the values for all the samples (dots) and in groups (boxes).

 
Alpha Diversity Box Plots for All Groups
 
 
 
 
 
 
 
Alpha Diversity Box Plots for Individual Comparisons
 
Comparison 1Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3View in PDFView in SVG
Comparison 2Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3View in PDFView in SVG
 
 
 

Group Significance of Alpha-diversity Indices

To test whether the alpha diversity among different comparison groups are different statisticall, we use the Kruskal Wallis H test provided the "alpha-group-significance" fucntion in the QIIME 2 "diversity" package. Kruskal Wallis H test is the non-parametric alternative to the One Way ANOVA. Non-parametric means that the test doesn’t assume your data comes from a particular distribution. The H test is used when the assumptions for ANOVA aren’t met (like the assumption of normality). It is sometimes called the one-way ANOVA on ranks, as the ranks of the data values are used in the test rather than the actual data points. The H test determines whether the medians of two or more groups are different.

Below are the Kruskal Wallis H test results for each comparison based on three different alpha diversity measures: 1) Observed species (features), 2) Shannon index, and 3) Simpson index.

 
 
Comparison 1.Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3Observed FeaturesShannon IndexSimpson Index
Comparison 2.Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3Observed FeaturesShannon IndexSimpson Index
 
 

IX. Analysis - Beta Diversity

 

NMDS and PCoA Plots

Beta diversity compares the similarity (or dissimilarity) of microbial profiles between different groups of samples. There are many different similarity/dissimilarity metrics. In general, they can be quantitative (using sequence abundance, e.g., Bray-Curtis or weighted UniFrac) or binary (considering only presence-absence of sequences, e.g., binary Jaccard or unweighted UniFrac). They can be even based on phylogeny (e.g., UniFrac metrics) or not (non-UniFrac metrics, such as Bray-Curtis, etc.).

For microbiome studies, species profiles of samples can be compared with the Bray-Curtis dissimilarity, which is based on the count data type. The pair-wise Bray-Curtis dissimilarity matrix of all samples can then be subject to either multi-dimensional scaling (MDS, also known as PCoA) or non-metric MDS (NMDS).

MDS/PCoA is a scaling or ordination method that starts with a matrix of similarities or dissimilarities between a set of samples and aims to produce a low-dimensional graphical plot of the data in such a way that distances between points in the plot are close to original dissimilarities.

NMDS is similar to MDS, however it does not use the dissimilarities data, instead it converts them into the ranks and use these ranks in the calculation.

In our beta diversity analysis, Bray-Curtis dissimilarity matrix was first calculated and then plotted by the PCoA and NMDS separately. Below are beta diveristy results for all groups together:

 
 
NMDS and PCoA Plots for All Groups
 
 
 
 
 

The above PCoA and NMDS plots are based on count data. The count data can also be transformed into centered log ratio (CLR) for each species. The CLR data is no longer count data and cannot be used in Bray-Curtis dissimilarity calculation. Instead CLR can be compared with Euclidean distances. When CLR data are compared by Euclidean distance, the distance is also called Aitchison distance.

Below are the NMDS and PCoA plots of the Aitchison distances of the samples:

 
 
 
 
 
 
 
NMDS and PCoA Plots for Individual Comparisons
 
 
Comparison No.Comparison NameNMDAPCoA
Bray-CurtisCLR EuclideanBray-CurtisCLR Euclidean
Comparison 1Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3PDFSVGPDFSVGPDFSVGPDFSVG
Comparison 2Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3PDFSVGPDFSVGPDFSVGPDFSVG
 
 
 
 
 

Interactive 3D PCoA Plots - Bray-Curtis Dissimilarity

 
 
 

Interactive 3D PCoA Plots - Euclidean Distance

 
 
 

Interactive 3D PCoA Plots - Correlation Coefficients

 
 
 

Group Significance of Beta-diversity Indices

To test whether the between-group dissimilarities are significantly greater than the within-group dissimilarities, the "beta-group-significance" function provided in the QIIME 2 "diversity" package was used with PERMANOVA (permutational multivariate analysis of variance) as the group significant testing method.

Three beta diversity matrics were used: 1) Bray–Curtis dissimilarity 2) Correlation coefficient matrix , and 3) Aitchison distance (Euclidean distance between clr-transformed compositions).

 
 
Comparison 1.Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3Bray–CurtisCorrelationAitchison
Comparison 2.Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3Bray–CurtisCorrelationAitchison
 
 
 

X. Analysis - Differential Abundance

16S rRNA next generation sequencing (NGS) generates a fixed number of reads that reflect the proportion of different species in a sample, i.e., the relative abundance of species, instead of the absolute abundance. In Mathematics, measurements involving probabilities, proportions, percentages, and ppm can all be thought of as compositional data. This makes the microbiome read count data “compositional” (Gloor et al, 2017). In general, compositional data represent parts of a whole which only carry relative information (http://www.compositionaldata.com/).

The problem of microbiome data being compositional arises when comparing two groups of samples for identifying “differentially abundant” species. A species with the same absolute abundance between two conditions, its relative abundances in the two conditions (e.g., percent abundance) can become different if the relative abundance of other species change greatly. This problem can lead to incorrect conclusion in terms of differential abundance for microbial species in the samples.

When studying differential abundance (DA), the current better approach is to transform the read count data into log ratio data. The ratios are calculated between read counts of all species in a sample to a “reference” count (e.g., mean read count of the sample). The log ratio data allow the detection of DA species without being affected by percentage bias mentioned above

In this report, a compositional DA analysis tool “ANCOM” (analysis of composition of microbiomes) was used. ANCOM transforms the count data into log-ratios and thus is more suitable for comparing the composition of microbiomes in two or more populations. "ANCOM" generates a table of features with W-statistics and whether the null hypothesis is rejected. The “W” is the W-statistic, or number of features that a single feature is tested to be significantly different against. Hence the higher the "W" the more statistical sifgnificane that a feature/species is differentially abundant.


References:

Gloor GB, Macklaim JM, Pawlowsky-Glahn V, Egozcue JJ. Microbiome Datasets Are Compositional: And This Is Not Optional. Front Microbiol. 2017 Nov 15;8:2224. doi: 10.3389/fmicb.2017.02224. PMID: 29187837; PMCID: PMC5695134.

Mandal S, Van Treuren W, White RA, Eggesbø M, Knight R, Peddada SD. Analysis of composition of microbiomes: a novel method for studying microbial composition. Microb Ecol Health Dis. 2015 May 29;26:27663. doi: 10.3402/mehd.v26.27663. PMID: 26028277; PMCID: PMC4450248.

Lin H, Peddada SD. Analysis of compositions of microbiomes with bias correction. Nat Commun. 2020 Jul 14;11(1):3514. doi: 10.1038/s41467-020-17041-7. PMID: 32665548; PMCID: PMC7360769.

 
 

ANCOM Differential Abundance Analysis

 
ANCOM Results for Individual Comparisons
Comparison No.Comparison Name
Comparison 1.Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3
Comparison 2.Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3
 
 

ANCOM-BC Differential Abundance Analysis

 

Starting with version V1.2, we also include the results of ANCOM-BC (Analysis of Compositions of Microbiomes with Bias Correction) (Lin and Peddada 2020). ANCOM-BC is an updated version of "ANCOM" that: (a) provides statistically valid test with appropriate p-values, (b) provides confidence intervals for differential abundance of each taxon, (c) controls the False Discovery Rate (FDR), (d) maintains adequate power, and (e) is computationally simple to implement. The bias correction (BC) addresses a challenging problem of the bias introduced by differences in the sampling fractions across samples. This bias has been a major hurdle in performing DA analysis of microbiome data. ANCOM-BC estimates the unknown sampling fractions and corrects the bias induced by their differences among samples. The absolute abundance data are modeled using a linear regression framework.

References:

Lin H, Peddada SD. Analysis of compositions of microbiomes with bias correction. Nat Commun. 2020 Jul 14;11(1):3514. doi: 10.1038/s41467-020-17041-7. PMID: 32665548; PMCID: PMC7360769.

 
 
ANCOM-BC Results for Individual Comparisons
 
Comparison No.Comparison Name
Comparison 1.Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3
Comparison 2.Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3
 
 
 

LEfSe - Linear Discriminant Analysis Effect Size

LEfSe (Linear Discriminant Analysis Effect Size) is an alternative method to find "organisms, genes, or pathways that consistently explain the differences between two or more microbial communities" (Segata et al., 2011). Specifically, LEfSe uses rank-based Kruskal-Wallis (KW) sum-rank test to detect features with significant differential (relative) abundance with respect to the class of interest. Since it is rank-based, instead of proportional based, the differential species identified among the comparison groups is less biased (than percent abundance based).

Reference:

Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, Huttenhower C. Metagenomic biomarker discovery and explanation. Genome Biol. 2011 Jun 24;12(6):R60. doi: 10.1186/gb-2011-12-6-r60. PMID: 21702898; PMCID: PMC3218848.

 
Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3
 
 
 
 
 
 
 
LEfSe Results for All Comparisons
 
Comparison No.Comparison Name
Comparison 1.Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3
Comparison 2.Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3
 
 

XI. Analysis - Heatmap Profile

 

Species vs Sample Abundance Heatmap for All Samples

 
 
 

Heatmaps for Individual Comparisons

 
A) Two-way clustering - clustered on both columns (Samples) and rows (organism)
Comparison No.Comparison NameFamily LevelGenus LevelSpecies Level
Comparison 1Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3PDFSVGPDFSVGPDFSVG
Comparison 2Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3PDFSVGPDFSVGPDFSVG
 
 
B) One-way clustering - clustered on rows (organism) only
Comparison No.Comparison NameFamily LevelGenus LevelSpecies Level
Comparison 1Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3PDFSVGPDFSVGPDFSVG
Comparison 2Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3PDFSVGPDFSVGPDFSVG
 
 
C) No clustering
Comparison No.Comparison NameFamily LevelGenus LevelSpecies Level
Comparison 1Saliva Visit 1 vs Saliva Visit 2 vs Saliva Visit 3PDFSVGPDFSVGPDFSVG
Comparison 2Plaque Visit 1 vs Plaque Visit 2 vs Plaque Visit 3PDFSVGPDFSVGPDFSVG
 
 

XII. Analysis - Network Association

To analyze the co-occurrence or co-exclusion between microbial species among different samples, network correlation analysis tools are usually used for this purpose. However, microbiome count data are compositional. If count data are normalized to the total number of counts in the sample, the data become not independent and traditional statistical metrics (e.g., correlation) for the detection of specie-species relationships can lead to spurious results. In addition, sequencing-based studies typically measure hundreds of OTUs (species) on few samples; thus, inference of OTU-OTU association networks is severely under-powered. Here we use SPIEC-EASI (SParse InversE Covariance Estimation for Ecological Association Inference), a statistical method for the inference of microbial ecological networks from amplicon sequencing datasets that addresses both of these issues (Kurtz et al., 2015). SPIEC-EASI combines data transformations developed for compositional data analysis with a graphical model inference framework that assumes the underlying ecological association network is sparse. SPIEC-EASI provides two algorithms for network inferencing – 1) Meinshausen-Bühlmann's neighborhood selection (MB method) and inverse covariance selection (GLASSO method, i.e., graphical least absolute shrinkage and selection operator). This is fundamentally distinct from SparCC, which essentially estimate pairwise correlations. In addition to these two methods, we provide the results of a third method - SparCC (Sparse Correlations for Compositional Data)(Friedman & Alm 2012), which is also a method for inferring correlations from compositional data. SparCC estimates the linear Pearson correlations between the log-transformed components.


References:

Kurtz ZD, Müller CL, Miraldi ER, Littman DR, Blaser MJ, Bonneau RA. Sparse and compositionally robust inference of microbial ecological networks. PLoS Comput Biol. 2015 May 7;11(5):e1004226. doi: 10.1371/journal.pcbi.1004226. PMID: 25950956; PMCID: PMC4423992.

Friedman J, Alm EJ. Inferring correlation networks from genomic survey data. PLoS Comput Biol. 2012;8(9):e1002687. doi: 10.1371/journal.pcbi.1002687. Epub 2012 Sep 20. PMID: 23028285; PMCID: PMC3447976.

 

SPIEC-EASI Network Inference by Neighborhood Selection (MB Method)

 
 

 

Association Network Inference by SparCC

 
 

 
 
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