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
XIII. Disclaimer

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FOMC Service Report

16S rRNA Gene V3V4 Amplicon Sequencing

Version V1.2

Version History

The Forsyth Institute, Cambridge, MA, USA
December 05, 2021

Project ID: FOMC0000

I. Project Summary

Project FOMC0000 services include NGS sequencing of the V3V4 region of the 16S rRNA 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


FOMC0000.S001100_2014zr0000_100V3V4_R1.fastq.gzzr0000_100V3V4_R2.fastq.gz


FOMC0000.S002101_2014zr0000_101V3V4_R1.fastq.gzzr0000_101V3V4_R2.fastq.gz


FOMC0000.S003102_2014zr0000_102V3V4_R1.fastq.gzzr0000_102V3V4_R2.fastq.gz


FOMC0000.S004103_2014zr0000_103V3V4_R1.fastq.gzzr0000_103V3V4_R2.fastq.gz


FOMC0000.S005104_2014zr0000_104V3V4_R1.fastq.gzzr0000_104V3V4_R2.fastq.gz


FOMC0000.S006107_2014zr0000_105V3V4_R1.fastq.gzzr0000_105V3V4_R2.fastq.gz


FOMC0000.S007108_2014zr0000_106V3V4_R1.fastq.gzzr0000_106V3V4_R2.fastq.gz


FOMC0000.S008110_2014zr0000_107V3V4_R1.fastq.gzzr0000_107V3V4_R2.fastq.gz


FOMC0000.S009112_2014zr0000_108V3V4_R1.fastq.gzzr0000_108V3V4_R2.fastq.gz


FOMC0000.S010113_2014zr0000_109V3V4_R1.fastq.gzzr0000_109V3V4_R2.fastq.gz


FOMC0000.S011114_2014zr0000_10V3V4_R1.fastq.gzzr0000_10V3V4_R2.fastq.gz


FOMC0000.S012115_2014zr0000_110V3V4_R1.fastq.gzzr0000_110V3V4_R2.fastq.gz


FOMC0000.S013119_2014zr0000_111V3V4_R1.fastq.gzzr0000_111V3V4_R2.fastq.gz


FOMC0000.S0141_2014zr0000_112V3V4_R1.fastq.gzzr0000_112V3V4_R2.fastq.gz


FOMC0000.S015120_2014zr0000_113V3V4_R1.fastq.gzzr0000_113V3V4_R2.fastq.gz


FOMC0000.S016121_2014zr0000_114V3V4_R1.fastq.gzzr0000_114V3V4_R2.fastq.gz


FOMC0000.S017122_2014zr0000_115V3V4_R1.fastq.gzzr0000_115V3V4_R2.fastq.gz


FOMC0000.S018123_2014zr0000_116V3V4_R1.fastq.gzzr0000_116V3V4_R2.fastq.gz


FOMC0000.S019130_2014zr0000_117V3V4_R1.fastq.gzzr0000_117V3V4_R2.fastq.gz


FOMC0000.S02013_2014zr0000_118V3V4_R1.fastq.gzzr0000_118V3V4_R2.fastq.gz


FOMC0000.S021132_2014zr0000_119V3V4_R1.fastq.gzzr0000_119V3V4_R2.fastq.gz


FOMC0000.S022133_2014zr0000_11V3V4_R1.fastq.gzzr0000_11V3V4_R2.fastq.gz


FOMC0000.S023134_2014zr0000_120V3V4_R1.fastq.gzzr0000_120V3V4_R2.fastq.gz


FOMC0000.S024136_2014zr0000_121V3V4_R1.fastq.gzzr0000_121V3V4_R2.fastq.gz


FOMC0000.S025139_2014zr0000_122V3V4_R1.fastq.gzzr0000_122V3V4_R2.fastq.gz


FOMC0000.S026140_2014zr0000_123V3V4_R1.fastq.gzzr0000_123V3V4_R2.fastq.gz


FOMC0000.S02714_2014zr0000_124V3V4_R1.fastq.gzzr0000_124V3V4_R2.fastq.gz


FOMC0000.S028142_2014zr0000_125V3V4_R1.fastq.gzzr0000_125V3V4_R2.fastq.gz


FOMC0000.S029146_2014zr0000_126V3V4_R1.fastq.gzzr0000_126V3V4_R2.fastq.gz


FOMC0000.S030148_2014zr0000_127V3V4_R1.fastq.gzzr0000_127V3V4_R2.fastq.gz


FOMC0000.S031149_2014zr0000_128V3V4_R1.fastq.gzzr0000_128V3V4_R2.fastq.gz


FOMC0000.S032150_2014zr0000_129V3V4_R1.fastq.gzzr0000_129V3V4_R2.fastq.gz


FOMC0000.S033159_2014zr0000_12V3V4_R1.fastq.gzzr0000_12V3V4_R2.fastq.gz


FOMC0000.S034161_2014zr0000_130V3V4_R1.fastq.gzzr0000_130V3V4_R2.fastq.gz


FOMC0000.S035162_2014zr0000_131V3V4_R1.fastq.gzzr0000_131V3V4_R2.fastq.gz


FOMC0000.S036164_2014zr0000_132V3V4_R1.fastq.gzzr0000_132V3V4_R2.fastq.gz


FOMC0000.S037168_2014zr0000_133V3V4_R1.fastq.gzzr0000_133V3V4_R2.fastq.gz


FOMC0000.S038170_2014zr0000_134V3V4_R1.fastq.gzzr0000_134V3V4_R2.fastq.gz


FOMC0000.S039171_2014zr0000_135V3V4_R1.fastq.gzzr0000_135V3V4_R2.fastq.gz


FOMC0000.S040176_2014zr0000_136V3V4_R1.fastq.gzzr0000_136V3V4_R2.fastq.gz


FOMC0000.S041177_2014zr0000_137V3V4_R1.fastq.gzzr0000_137V3V4_R2.fastq.gz


FOMC0000.S042180_2014zr0000_138V3V4_R1.fastq.gzzr0000_138V3V4_R2.fastq.gz


FOMC0000.S043187_2014zr0000_139V3V4_R1.fastq.gzzr0000_139V3V4_R2.fastq.gz


FOMC0000.S044188_2014zr0000_13V3V4_R1.fastq.gzzr0000_13V3V4_R2.fastq.gz


FOMC0000.S045189_2014zr0000_140V3V4_R1.fastq.gzzr0000_140V3V4_R2.fastq.gz


FOMC0000.S046191_2014zr0000_141V3V4_R1.fastq.gzzr0000_141V3V4_R2.fastq.gz


FOMC0000.S047193_2014zr0000_142V3V4_R1.fastq.gzzr0000_142V3V4_R2.fastq.gz


FOMC0000.S048196_2014zr0000_143V3V4_R1.fastq.gzzr0000_143V3V4_R2.fastq.gz


FOMC0000.S049198_2014zr0000_144V3V4_R1.fastq.gzzr0000_144V3V4_R2.fastq.gz


FOMC0000.S050200_2014zr0000_145V3V4_R1.fastq.gzzr0000_145V3V4_R2.fastq.gz


FOMC0000.S051201_2014zr0000_146V3V4_R1.fastq.gzzr0000_146V3V4_R2.fastq.gz


FOMC0000.S052204_2014zr0000_147V3V4_R1.fastq.gzzr0000_147V3V4_R2.fastq.gz


FOMC0000.S053205_2014zr0000_148V3V4_R1.fastq.gzzr0000_148V3V4_R2.fastq.gz


FOMC0000.S054207_2014zr0000_149V3V4_R1.fastq.gzzr0000_149V3V4_R2.fastq.gz


FOMC0000.S055208_2014zr0000_14V3V4_R1.fastq.gzzr0000_14V3V4_R2.fastq.gz


FOMC0000.S056209_2014zr0000_150V3V4_R1.fastq.gzzr0000_150V3V4_R2.fastq.gz


FOMC0000.S05721_2014zr0000_151V3V4_R1.fastq.gzzr0000_151V3V4_R2.fastq.gz


FOMC0000.S058214_2014zr0000_152V3V4_R1.fastq.gzzr0000_152V3V4_R2.fastq.gz


FOMC0000.S059215_2014zr0000_153V3V4_R1.fastq.gzzr0000_153V3V4_R2.fastq.gz


FOMC0000.S060216_2014zr0000_15V3V4_R1.fastq.gzzr0000_15V3V4_R2.fastq.gz


FOMC0000.S061217_2014zr0000_16V3V4_R1.fastq.gzzr0000_16V3V4_R2.fastq.gz


FOMC0000.S062218_2014zr0000_17V3V4_R1.fastq.gzzr0000_17V3V4_R2.fastq.gz


FOMC0000.S063219_2014zr0000_18V3V4_R1.fastq.gzzr0000_18V3V4_R2.fastq.gz


FOMC0000.S064220_2014zr0000_19V3V4_R1.fastq.gzzr0000_19V3V4_R2.fastq.gz


FOMC0000.S065221_2014zr0000_1V3V4_R1.fastq.gzzr0000_1V3V4_R2.fastq.gz


FOMC0000.S066225_2014zr0000_20V3V4_R1.fastq.gzzr0000_20V3V4_R2.fastq.gz


FOMC0000.S067234_2014zr0000_21V3V4_R1.fastq.gzzr0000_21V3V4_R2.fastq.gz


FOMC0000.S068240_2014zr0000_22V3V4_R1.fastq.gzzr0000_22V3V4_R2.fastq.gz


FOMC0000.S069243_2014zr0000_23V3V4_R1.fastq.gzzr0000_23V3V4_R2.fastq.gz


FOMC0000.S070246_2014zr0000_24V3V4_R1.fastq.gzzr0000_24V3V4_R2.fastq.gz


FOMC0000.S071251_2014zr0000_25V3V4_R1.fastq.gzzr0000_25V3V4_R2.fastq.gz


FOMC0000.S072259_2014zr0000_26V3V4_R1.fastq.gzzr0000_26V3V4_R2.fastq.gz


FOMC0000.S073263_2014zr0000_27V3V4_R1.fastq.gzzr0000_27V3V4_R2.fastq.gz


FOMC0000.S074276_2014zr0000_28V3V4_R1.fastq.gzzr0000_28V3V4_R2.fastq.gz


FOMC0000.S075287_2014zr0000_29V3V4_R1.fastq.gzzr0000_29V3V4_R2.fastq.gz


FOMC0000.S076288_2014zr0000_2V3V4_R1.fastq.gzzr0000_2V3V4_R2.fastq.gz


FOMC0000.S077293_2014zr0000_30V3V4_R1.fastq.gzzr0000_30V3V4_R2.fastq.gz


FOMC0000.S078295_2014zr0000_31V3V4_R1.fastq.gzzr0000_31V3V4_R2.fastq.gz


FOMC0000.S0793_2014zr0000_32V3V4_R1.fastq.gzzr0000_32V3V4_R2.fastq.gz


FOMC0000.S08072_2014zr0000_33V3V4_R1.fastq.gzzr0000_33V3V4_R2.fastq.gz


FOMC0000.S08175_2014zr0000_34V3V4_R1.fastq.gzzr0000_34V3V4_R2.fastq.gz


FOMC0000.S08276_2014zr0000_35V3V4_R1.fastq.gzzr0000_35V3V4_R2.fastq.gz


FOMC0000.S08377_2014zr0000_36V3V4_R1.fastq.gzzr0000_36V3V4_R2.fastq.gz


FOMC0000.S08478_2014zr0000_37V3V4_R1.fastq.gzzr0000_37V3V4_R2.fastq.gz


FOMC0000.S08580_2014zr0000_38V3V4_R1.fastq.gzzr0000_38V3V4_R2.fastq.gz


FOMC0000.S08687_2014zr0000_39V3V4_R1.fastq.gzzr0000_39V3V4_R2.fastq.gz


FOMC0000.S08788_2014zr0000_3V3V4_R1.fastq.gzzr0000_3V3V4_R2.fastq.gz


FOMC0000.S08892_2014zr0000_40V3V4_R1.fastq.gzzr0000_40V3V4_R2.fastq.gz


FOMC0000.S08993_2014zr0000_41V3V4_R1.fastq.gzzr0000_41V3V4_R2.fastq.gz


FOMC0000.S09094_2014zr0000_42V3V4_R1.fastq.gzzr0000_42V3V4_R2.fastq.gz


FOMC0000.S09195_2014zr0000_43V3V4_R1.fastq.gzzr0000_43V3V4_R2.fastq.gz


FOMC0000.S09297_2014zr0000_44V3V4_R1.fastq.gzzr0000_44V3V4_R2.fastq.gz


FOMC0000.S09398_2014zr0000_45V3V4_R1.fastq.gzzr0000_45V3V4_R2.fastq.gz


FOMC0000.S09499_2014zr0000_46V3V4_R1.fastq.gzzr0000_46V3V4_R2.fastq.gz


FOMC0000.S095105_2016zr0000_47V3V4_R1.fastq.gzzr0000_47V3V4_R2.fastq.gz


FOMC0000.S096135_2016zr0000_48V3V4_R1.fastq.gzzr0000_48V3V4_R2.fastq.gz


FOMC0000.S097138_2016zr0000_49V3V4_R1.fastq.gzzr0000_49V3V4_R2.fastq.gz


FOMC0000.S098141_2016zr0000_4V3V4_R1.fastq.gzzr0000_4V3V4_R2.fastq.gz


FOMC0000.S099143_2016zr0000_50V3V4_R1.fastq.gzzr0000_50V3V4_R2.fastq.gz


FOMC0000.S100144_2016zr0000_51V3V4_R1.fastq.gzzr0000_51V3V4_R2.fastq.gz


FOMC0000.S101147_2016zr0000_52V3V4_R1.fastq.gzzr0000_52V3V4_R2.fastq.gz


FOMC0000.S102153_2016zr0000_53V3V4_R1.fastq.gzzr0000_53V3V4_R2.fastq.gz


FOMC0000.S103154_2016zr0000_54V3V4_R1.fastq.gzzr0000_54V3V4_R2.fastq.gz


FOMC0000.S104155_2016zr0000_55V3V4_R1.fastq.gzzr0000_55V3V4_R2.fastq.gz


FOMC0000.S105156_2016zr0000_56V3V4_R1.fastq.gzzr0000_56V3V4_R2.fastq.gz


FOMC0000.S106157_2016zr0000_57V3V4_R1.fastq.gzzr0000_57V3V4_R2.fastq.gz


FOMC0000.S107158_2016zr0000_58V3V4_R1.fastq.gzzr0000_58V3V4_R2.fastq.gz


FOMC0000.S108163_2016zr0000_59V3V4_R1.fastq.gzzr0000_59V3V4_R2.fastq.gz


FOMC0000.S109167_2016zr0000_5V3V4_R1.fastq.gzzr0000_5V3V4_R2.fastq.gz


FOMC0000.S110169_2016zr0000_60V3V4_R1.fastq.gzzr0000_60V3V4_R2.fastq.gz


FOMC0000.S111173_2016zr0000_61V3V4_R1.fastq.gzzr0000_61V3V4_R2.fastq.gz


FOMC0000.S112175_2016zr0000_62V3V4_R1.fastq.gzzr0000_62V3V4_R2.fastq.gz


FOMC0000.S113178_2016zr0000_63V3V4_R1.fastq.gzzr0000_63V3V4_R2.fastq.gz


FOMC0000.S114179_2016zr0000_64V3V4_R1.fastq.gzzr0000_64V3V4_R2.fastq.gz


FOMC0000.S115181_2016zr0000_65V3V4_R1.fastq.gzzr0000_65V3V4_R2.fastq.gz


FOMC0000.S116182_2016zr0000_66V3V4_R1.fastq.gzzr0000_66V3V4_R2.fastq.gz


FOMC0000.S117183_2016zr0000_67V3V4_R1.fastq.gzzr0000_67V3V4_R2.fastq.gz


FOMC0000.S118184_2016zr0000_68V3V4_R1.fastq.gzzr0000_68V3V4_R2.fastq.gz


FOMC0000.S119185_2016zr0000_69V3V4_R1.fastq.gzzr0000_69V3V4_R2.fastq.gz


FOMC0000.S120192_2016zr0000_6V3V4_R1.fastq.gzzr0000_6V3V4_R2.fastq.gz


FOMC0000.S121194_2016zr0000_70V3V4_R1.fastq.gzzr0000_70V3V4_R2.fastq.gz


FOMC0000.S122195_2016zr0000_71V3V4_R1.fastq.gzzr0000_71V3V4_R2.fastq.gz


FOMC0000.S123197_2016zr0000_72V3V4_R1.fastq.gzzr0000_72V3V4_R2.fastq.gz


FOMC0000.S124199_2016zr0000_73V3V4_R1.fastq.gzzr0000_73V3V4_R2.fastq.gz


FOMC0000.S125200_2016zr0000_74V3V4_R1.fastq.gzzr0000_74V3V4_R2.fastq.gz


FOMC0000.S126206_2016zr0000_75V3V4_R1.fastq.gzzr0000_75V3V4_R2.fastq.gz


FOMC0000.S127210_2016zr0000_76V3V4_R1.fastq.gzzr0000_76V3V4_R2.fastq.gz


FOMC0000.S128211_2016zr0000_77V3V4_R1.fastq.gzzr0000_77V3V4_R2.fastq.gz


FOMC0000.S129212_2016zr0000_78V3V4_R1.fastq.gzzr0000_78V3V4_R2.fastq.gz


FOMC0000.S130213_2016zr0000_79V3V4_R1.fastq.gzzr0000_79V3V4_R2.fastq.gz


FOMC0000.S131222_2016zr0000_7V3V4_R1.fastq.gzzr0000_7V3V4_R2.fastq.gz


FOMC0000.S132227_2016zr0000_80V3V4_R1.fastq.gzzr0000_80V3V4_R2.fastq.gz


FOMC0000.S133228_2016zr0000_81V3V4_R1.fastq.gzzr0000_81V3V4_R2.fastq.gz


FOMC0000.S134230_2016zr0000_82V3V4_R1.fastq.gzzr0000_82V3V4_R2.fastq.gz


FOMC0000.S135232_2016zr0000_83V3V4_R1.fastq.gzzr0000_83V3V4_R2.fastq.gz


FOMC0000.S136235_2016zr0000_84V3V4_R1.fastq.gzzr0000_84V3V4_R2.fastq.gz


FOMC0000.S137236_2016zr0000_85V3V4_R1.fastq.gzzr0000_85V3V4_R2.fastq.gz


FOMC0000.S138238_2016zr0000_86V3V4_R1.fastq.gzzr0000_86V3V4_R2.fastq.gz


FOMC0000.S139261_2016zr0000_87V3V4_R1.fastq.gzzr0000_87V3V4_R2.fastq.gz


FOMC0000.S140271_2016zr0000_88V3V4_R1.fastq.gzzr0000_88V3V4_R2.fastq.gz


FOMC0000.S141277_2016zr0000_89V3V4_R1.fastq.gzzr0000_89V3V4_R2.fastq.gz


FOMC0000.S142284_2016zr0000_8V3V4_R1.fastq.gzzr0000_8V3V4_R2.fastq.gz


FOMC0000.S143285_2016zr0000_90V3V4_R1.fastq.gzzr0000_90V3V4_R2.fastq.gz


FOMC0000.S14441_2016zr0000_91V3V4_R1.fastq.gzzr0000_91V3V4_R2.fastq.gz


FOMC0000.S14573_2016zr0000_92V3V4_R1.fastq.gzzr0000_92V3V4_R2.fastq.gz


FOMC0000.S14674_2016zr0000_93V3V4_R1.fastq.gzzr0000_93V3V4_R2.fastq.gz


FOMC0000.S14779_2016zr0000_94V3V4_R1.fastq.gzzr0000_94V3V4_R2.fastq.gz


FOMC0000.S14881_2016zr0000_95V3V4_R1.fastq.gzzr0000_95V3V4_R2.fastq.gz


FOMC0000.S14982_2016zr0000_96V3V4_R1.fastq.gzzr0000_96V3V4_R2.fastq.gz


FOMC0000.S15083_2016zr0000_97V3V4_R1.fastq.gzzr0000_97V3V4_R2.fastq.gz


FOMC0000.S15184_2016zr0000_98V3V4_R1.fastq.gzzr0000_98V3V4_R2.fastq.gz


FOMC0000.S15290_2016zr0000_99V3V4_R1.fastq.gzzr0000_99V3V4_R2.fastq.gz


FOMC0000.S15391_2016zr0000_9V3V4_R1.fastq.gzzr0000_9V3V4_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”.

Below is the link to a PDF file for viewing the 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/R2251241231221211201
25056.07%66.11%70.19%70.57%70.67%71.21%
24056.37%66.65%70.80%71.16%71.27%71.77%
23056.81%67.73%71.70%71.99%72.04%23.38%
22057.45%68.46%72.44%72.87%23.33%21.44%
21058.07%69.01%73.05%23.67%21.73%0.00%

Based on the above result, the trim length combination of R1 = 210 bases and R2 = 231 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 IDF0000.S001F0000.S002F0000.S003F0000.S004F0000.S005F0000.S006F0000.S007F0000.S008F0000.S009F0000.S010F0000.S011F0000.S012F0000.S013F0000.S014F0000.S015F0000.S016F0000.S017F0000.S018F0000.S019F0000.S020F0000.S021F0000.S022F0000.S023F0000.S024F0000.S025F0000.S026F0000.S027F0000.S028F0000.S029F0000.S030F0000.S031F0000.S032F0000.S033F0000.S034F0000.S035F0000.S036F0000.S037F0000.S038F0000.S039F0000.S040F0000.S041F0000.S042F0000.S043F0000.S044F0000.S045F0000.S046F0000.S047F0000.S048F0000.S049F0000.S050F0000.S051F0000.S052F0000.S053F0000.S054F0000.S055F0000.S056F0000.S057F0000.S058F0000.S059F0000.S060F0000.S061F0000.S062F0000.S063F0000.S064F0000.S065F0000.S066F0000.S067F0000.S068F0000.S069F0000.S070F0000.S071F0000.S072F0000.S073F0000.S074F0000.S075F0000.S076F0000.S077F0000.S078F0000.S079F0000.S080F0000.S081F0000.S082F0000.S083F0000.S084F0000.S085F0000.S086F0000.S087F0000.S088F0000.S089F0000.S090F0000.S091F0000.S092F0000.S093F0000.S094F0000.S095F0000.S096F0000.S097F0000.S098F0000.S099F0000.S100F0000.S101F0000.S102F0000.S103F0000.S104F0000.S105F0000.S106F0000.S107F0000.S108F0000.S109F0000.S110F0000.S111F0000.S112F0000.S113F0000.S114F0000.S115F0000.S116F0000.S117F0000.S118F0000.S119F0000.S120F0000.S121F0000.S122F0000.S123F0000.S124F0000.S125F0000.S126F0000.S127F0000.S128F0000.S129F0000.S130F0000.S131F0000.S132F0000.S133F0000.S134F0000.S135F0000.S136F0000.S137F0000.S138F0000.S139F0000.S140F0000.S141F0000.S142F0000.S143F0000.S144F0000.S145F0000.S146F0000.S147F0000.S148F0000.S149F0000.S150F0000.S151F0000.S152F0000.S153Row SumPercentage
input137,933158,464116,599128,07152,072127,997145,498108,32398,34687,499131,709127,676112,314158,078124,537125,332139,907140,357107,190103,439148,030168,282105,451113,38664,332153,16858,341136,918138,524118,186110,379118,624139,383143,666136,998105,086144,982164,653125,276127,010129,689179,172115,022124,791113,092107,250113,25388,07499,161136,95380,032131,329132,947115,598108,28595,06292,431122,725123,696125,729120,843132,56386,582114,212107,78487,26988,141120,151142,10789,69688,665142,115167,673201,920188,678173,706137,090163,27163,333162,834109,716283,113169,976114,580734149,709123,67578,24096,890176,746218,963172,152128,071248,827125,164119,205147,826181,290110,607120,027201,314116,175129,502118,217141,75986,128102,658140,308180,923128,955118,115144,271137,654122,178144,983126,917114,673105,46795,144155,442165,991181,54879,417141,017101,10792,5973,787133,829113,750128,885207,046251,69893,58992,409101,117119,830116,901110,04469,591146,038264,380180,835126,758150,76884,78475,980133,941109,177124,54599,952153,251116,732174,67419,597,202100.00%
filtered123,904146,028106,933117,80448,385121,079132,292101,51292,09381,263126,756115,865101,306149,814113,734114,934130,279129,839100,22895,621135,275160,15999,719107,01160,291141,36354,079124,507130,676109,538101,512110,304135,343129,899122,10295,885133,760151,098116,197119,147118,368163,490106,484120,756104,63797,637103,90482,34991,424128,27174,145118,898125,310105,467105,83990,46986,286115,394113,649120,980113,452127,34183,770111,427103,74484,06684,589115,590137,36786,34785,360137,536162,004195,431184,054167,474132,215158,93161,200156,305106,022272,403160,519111,118615144,269118,66573,00993,219170,876212,127164,557123,806240,514121,088116,146142,873175,084105,939114,363196,453112,045125,728113,833136,92383,43899,348136,095174,797123,906113,430137,893132,682113,816140,020121,877110,194101,45991,396149,071161,340174,77176,448136,53298,66489,4133,490129,205110,368123,722199,772243,55090,16889,11397,382110,959113,360106,85067,677141,878256,534175,999121,845145,50582,36373,673128,640102,425114,41093,167140,266109,164170,17018,609,93394.96%
denoisedF120,426143,774104,842114,75247,355119,873128,56799,14790,73179,598123,847113,57298,656148,329110,323113,272128,000127,12498,37493,620131,136156,78097,934104,98459,214138,12452,812121,738129,110107,27299,294107,594133,040126,392118,01493,374130,000148,388114,298117,824115,017159,729103,857118,986102,47594,629101,47381,01689,715126,80372,656115,533123,198103,308104,20389,32384,619113,899110,641119,401111,962125,18182,573109,725100,89782,87682,670113,157136,12084,28083,727135,475160,103194,004182,330165,255129,747156,37959,812153,118104,524267,388158,880109,464543141,635115,62571,42791,185169,815209,226161,856121,487236,595119,610114,345140,437172,665103,546111,691194,537110,188124,137112,028134,56282,00697,495133,971171,810122,204110,318135,338130,803110,033137,766119,542107,93899,72689,496147,216159,176171,21775,423134,51197,84587,5713,395126,861107,989120,504195,295241,88088,21787,53996,078108,443111,907105,58266,698139,909250,844173,174119,220143,46681,27772,515126,190100,649112,18891,373138,021107,438167,75718,274,58693.25%
denoisedR115,429136,212102,127110,13945,559117,272123,86995,70888,86177,344122,241110,25495,008144,019104,861110,396124,654123,47895,64290,827127,238154,43994,229102,02256,724133,84650,924116,998125,049103,12996,603104,009131,349121,537113,87090,219126,086144,004111,676115,311111,121155,805100,275117,94199,15491,14998,77278,24086,566123,96870,458110,381118,64999,667102,71787,16581,955111,099106,688118,388110,329123,88381,918107,956100,40081,71081,160108,226134,58983,03582,298134,127158,123191,916180,243163,137128,289153,15958,845150,568103,195263,039157,174107,331528139,047113,57370,41389,021168,095206,532159,218120,150233,797118,160113,055138,568171,083101,322109,315191,373108,043123,415110,878132,42680,94495,746131,999169,983118,946107,465132,539128,649107,564135,866117,527106,04698,68388,276145,825156,566169,07374,571132,92296,81485,9483,301124,378106,354119,545190,911237,98187,21885,41995,302107,145110,428104,39765,623138,966249,610170,806117,497141,80080,51671,669123,87397,730109,31089,155133,648104,119165,61617,900,25191.34%
merged101,484125,06391,81098,13541,546109,830112,55987,28381,27770,946115,79899,65285,085133,70393,51599,718113,721112,04086,79981,830113,091144,54184,58791,75550,288123,73346,392106,892118,90593,29987,93692,079121,548109,160100,47281,552115,624134,012102,068106,61399,721146,77092,087110,97491,00284,33387,61174,67578,035113,06263,203101,459111,44893,40097,21480,24375,784103,01198,530110,455103,351114,97176,46499,92296,94278,22973,751102,043126,91475,34075,409126,123155,588186,381170,041160,600119,537143,19854,332142,80095,161244,799154,14199,822440130,079106,26865,13082,124160,187193,559147,951111,105224,199110,075106,063131,739162,43994,112101,760188,830100,027114,727101,677123,54474,24888,361124,399151,706111,84498,940122,574120,56696,875127,159109,16296,53291,14980,203138,578144,087156,66569,753123,66992,80478,5213,092114,19797,821115,238185,048233,71679,53077,91791,847102,168104,55398,85461,706129,806242,205166,563110,660138,84075,60765,992116,34089,77997,64280,813124,42993,517162,03316,668,96085.06%
nonchim70,408104,05072,25866,39435,24084,18492,45663,45057,80752,72389,71479,04760,901107,60173,44377,91882,86070,84764,25462,28287,073106,04860,64670,96038,982105,82435,95377,951105,71568,34063,46170,22492,21984,69767,98562,05588,996110,97980,15982,61074,379127,53464,83176,52868,98573,43661,42671,04761,83384,94748,55575,66996,07781,51172,31161,86061,59682,34780,29691,66078,44485,89157,82277,15492,75270,76451,55779,936101,32758,21461,07397,955153,316162,314134,382157,89081,143113,49940,399115,38970,101176,840149,28673,95935487,42286,10648,24468,094106,226141,493107,89481,076166,91481,91175,484114,168130,37769,73477,578186,16471,20476,62773,99596,65959,76164,95798,236128,65886,01173,59695,25290,24371,808102,67284,63367,22166,45858,253114,351106,573126,02153,85795,07770,81160,5582,85581,29372,071109,533174,703226,94855,66059,63779,44881,11180,53177,68545,71399,157229,989162,43089,553132,51959,80249,98589,15255,36867,39762,27793,08776,893159,18513,183,66267.27%

This table can be downloaded as an Excel table below:

 

5. DADA2 Amplicon Sequence Variants (ASVs). A total of 6443 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

#SampleIDVisit_IDYearRunCavities_1_01_Gender28_FluorideProductssubgroupcompGroupcolorGroup##subGroup:all;compGroup:Cavity_vs_NoCavity
F0000.S001100_20142014010allNoCavityCavityF0000.S001
F0000.S002101_20142014111allCavityCavityF0000.S002
F0000.S003102_20142014000allNoCavityNoCavityF0000.S003
F0000.S004103_20142014010allNoCavityCavityF0000.S004
F0000.S005104_20142014000allNoCavityNoCavityF0000.S005
F0000.S006107_20142014110allCavityCavityF0000.S006
F0000.S007108_20142014010allNoCavityCavityF0000.S007
F0000.S008110_20142014001allNoCavityNoCavityF0000.S008
F0000.S009112_20142014001allNoCavityNoCavityF0000.S009
F0000.S010113_20142014001allNoCavityNoCavityF0000.S010
F0000.S011114_20142014110allCavityCavityF0000.S011
F0000.S012115_20142014010allNoCavityCavityF0000.S012
F0000.S013119_20142014011allNoCavityCavityF0000.S013
F0000.S0141_20142014000allNoCavityNoCavityF0000.S014
F0000.S015120_20142014000allNoCavityNoCavityF0000.S015
F0000.S016121_20142014111allCavityCavityF0000.S016
F0000.S017122_20142014001allNoCavityNoCavityF0000.S017
F0000.S018123_20142014011allNoCavityCavityF0000.S018
F0000.S019130_20142014101allCavityNoCavityF0000.S019
F0000.S02013_20142014110allCavityCavityF0000.S020
F0000.S021132_20142014210allCavityCavityF0000.S021
F0000.S022133_20142014011allNoCavityCavityF0000.S022
F0000.S023134_20142014100allCavityNoCavityF0000.S023
F0000.S024136_20142014000allNoCavityNoCavityF0000.S024
F0000.S025139_20142014010allNoCavityCavityF0000.S025
F0000.S026140_20142014011allNoCavityCavityF0000.S026
F0000.S02714_2014201410NAallCavityNoCavityF0000.S027
F0000.S028142_20142014110allCavityCavityF0000.S028
F0000.S029146_20142014000allNoCavityNoCavityF0000.S029
F0000.S030148_20142014011allNoCavityCavityF0000.S030
F0000.S031149_20142014000allNoCavityNoCavityF0000.S031
F0000.S032150_20142014100allCavityNoCavityF0000.S032
F0000.S033159_20142014010allNoCavityCavityF0000.S033
F0000.S034161_20142014000allNoCavityNoCavityF0000.S034
F0000.S035162_20142014010allNoCavityCavityF0000.S035
F0000.S036164_20142014100allCavityNoCavityF0000.S036
F0000.S037168_20142014100allCavityNoCavityF0000.S037
F0000.S038170_20142014001allNoCavityNoCavityF0000.S038
F0000.S039171_20142014010allNoCavityCavityF0000.S039
F0000.S040176_20142014000allNoCavityNoCavityF0000.S040
F0000.S041177_20142014101allCavityNoCavityF0000.S041
F0000.S042180_20142014101allCavityNoCavityF0000.S042
F0000.S043187_20142014010allNoCavityCavityF0000.S043
F0000.S044188_20142014010allNoCavityCavityF0000.S044
F0000.S045189_20142014100allCavityNoCavityF0000.S045
F0000.S046191_20142014001allNoCavityNoCavityF0000.S046
F0000.S047193_20142014010allNoCavityCavityF0000.S047
F0000.S048196_20142014110allCavityCavityF0000.S048
F0000.S049198_20142014110allCavityCavityF0000.S049
F0000.S050200_20142014010allNoCavityCavityF0000.S050
F0000.S051201_20142014000allNoCavityNoCavityF0000.S051
F0000.S052204_20142014010allNoCavityCavityF0000.S052
F0000.S053205_20142014010allNoCavityCavityF0000.S053
F0000.S054207_20142014101allCavityNoCavityF0000.S054
F0000.S055208_20142014011allNoCavityCavityF0000.S055
F0000.S056209_20142014000allNoCavityNoCavityF0000.S056
F0000.S05721_20142014111allCavityCavityF0000.S057
F0000.S058214_20142014011allNoCavityCavityF0000.S058
F0000.S059215_20142014010allNoCavityCavityF0000.S059
F0000.S060216_20142014010allNoCavityCavityF0000.S060
F0000.S061217_20142014010allNoCavityCavityF0000.S061
F0000.S062218_20142014110allCavityCavityF0000.S062
F0000.S063219_20142014101allCavityNoCavityF0000.S063
F0000.S064220_20142014111allCavityCavityF0000.S064
F0000.S065221_20142014010allNoCavityCavityF0000.S065
F0000.S066225_20142014011allNoCavityCavityF0000.S066
F0000.S067234_20142014011allNoCavityCavityF0000.S067
F0000.S068240_20142014101allCavityNoCavityF0000.S068
F0000.S069243_20142014000allNoCavityNoCavityF0000.S069
F0000.S070246_20142014010allNoCavityCavityF0000.S070
F0000.S071251_20142014011allNoCavityCavityF0000.S071
F0000.S072259_20142014001allNoCavityNoCavityF0000.S072
F0000.S073263_20142014101allCavityNoCavityF0000.S073
F0000.S074276_20142014000allNoCavityNoCavityF0000.S074
F0000.S075287_20142014001allNoCavityNoCavityF0000.S075
F0000.S076288_20142014110allCavityCavityF0000.S076
F0000.S077293_20142014110allCavityCavityF0000.S077
F0000.S078295_20142014000allNoCavityNoCavityF0000.S078
F0000.S0793_20142014011allNoCavityCavityF0000.S079
F0000.S08072_20142014001allNoCavityNoCavityF0000.S080
F0000.S08175_20142014111allCavityCavityF0000.S081
F0000.S08276_20142014111allCavityCavityF0000.S082
F0000.S08377_20142014011allNoCavityCavityF0000.S083
F0000.S08478_20142014010allNoCavityCavityF0000.S084
F0000.S08580_20142014011allNoCavityCavityF0000.S085
F0000.S08687_20142014100allCavityNoCavityF0000.S086
F0000.S08788_20142014010allNoCavityCavityF0000.S087
F0000.S08892_20142014000allNoCavityNoCavityF0000.S088
F0000.S08993_20142014011allNoCavityCavityF0000.S089
F0000.S09094_20142014101allCavityNoCavityF0000.S090
F0000.S09195_20142014110allCavityCavityF0000.S091
F0000.S09297_20142014110allCavityCavityF0000.S092
F0000.S09398_20142014001allNoCavityNoCavityF0000.S093
F0000.S09499_20142014000allNoCavityNoCavityF0000.S094
F0000.S095105_20162016100allCavityNoCavityF0000.S095
F0000.S096135_20162016110allCavityCavityF0000.S096
F0000.S097138_20162016110allCavityCavityF0000.S097
F0000.S098141_20162016110allCavityCavityF0000.S098
F0000.S099143_20162016010allNoCavityCavityF0000.S099
F0000.S100144_20162016101allCavityNoCavityF0000.S100
F0000.S101147_20162016001allNoCavityNoCavityF0000.S101
F0000.S102153_20162016110allCavityCavityF0000.S102
F0000.S103154_20162016001allNoCavityNoCavityF0000.S103
F0000.S104155_20162016100allCavityNoCavityF0000.S104
F0000.S105156_20162016000allNoCavityNoCavityF0000.S105
F0000.S106157_20162016000allNoCavityNoCavityF0000.S106
F0000.S107158_20162016001allNoCavityNoCavityF0000.S107
F0000.S108163_20162016001allNoCavityNoCavityF0000.S108
F0000.S109167_20162016001allNoCavityNoCavityF0000.S109
F0000.S110169_20162016010allNoCavityCavityF0000.S110
F0000.S111173_20162016110allCavityCavityF0000.S111
F0000.S112175_20162016010allNoCavityCavityF0000.S112
F0000.S113178_20162016010allNoCavityCavityF0000.S113
F0000.S114179_20162016001allNoCavityNoCavityF0000.S114
F0000.S115181_20162016001allNoCavityNoCavityF0000.S115
F0000.S116182_20162016111allCavityCavityF0000.S116
F0000.S117183_20162016000allNoCavityNoCavityF0000.S117
F0000.S118184_20162016001allNoCavityNoCavityF0000.S118
F0000.S119185_20162016100allCavityNoCavityF0000.S119
F0000.S120192_20162016110allCavityCavityF0000.S120
F0000.S121194_20162016100allCavityNoCavityF0000.S121
F0000.S122195_20162016101allCavityNoCavityF0000.S122
F0000.S123197_20162016010allNoCavityCavityF0000.S123
F0000.S124199_20162016010allNoCavityCavityF0000.S124
F0000.S125200_20162016110allCavityCavityF0000.S125
F0000.S126206_20162016110allCavityCavityF0000.S126
F0000.S127210_20162016000allNoCavityNoCavityF0000.S127
F0000.S128211_20162016101allCavityNoCavityF0000.S128
F0000.S129212_20162016100allCavityNoCavityF0000.S129
F0000.S130213_20162016110allCavityCavityF0000.S130
F0000.S131222_20162016010allNoCavityCavityF0000.S131
F0000.S132227_20162016100allCavityNoCavityF0000.S132
F0000.S133228_20162016110allCavityCavityF0000.S133
F0000.S134230_20162016010allNoCavityCavityF0000.S134
F0000.S135232_20162016011allNoCavityCavityF0000.S135
F0000.S136235_20162016100allCavityNoCavityF0000.S136
F0000.S137236_20162016000allNoCavityNoCavityF0000.S137
F0000.S138238_20162016010allNoCavityCavityF0000.S138
F0000.S139261_20162016111allCavityCavityF0000.S139
F0000.S140271_20162016010allNoCavityCavityF0000.S140
F0000.S141277_20162016110allCavityCavityF0000.S141
F0000.S142284_20162016010allNoCavityCavityF0000.S142
F0000.S143285_20162016000allNoCavityNoCavityF0000.S143
F0000.S14441_20162016000allNoCavityNoCavityF0000.S144
F0000.S14573_20162016010allNoCavityCavityF0000.S145
F0000.S14674_20162016010allNoCavityCavityF0000.S146
F0000.S14779_20162016010allNoCavityCavityF0000.S147
F0000.S14881_20162016000allNoCavityNoCavityF0000.S148
F0000.S14982_20162016010allNoCavityCavityF0000.S149
F0000.S15083_20162016000allNoCavityNoCavityF0000.S150
F0000.S15184_20162016000allNoCavityNoCavityF0000.S151
F0000.S15290_20162016000allNoCavityNoCavityF0000.S152
F0000.S15391_20162016110allCavityCavityF0000.S153
 
 

ASV Read Counts by Samples

#Sample IDRead Count
F0000.S085354
F0000.S1272,855
F0000.S00535,240
F0000.S02735,953
F0000.S02538,982
F0000.S07940,399
F0000.S13945,713
F0000.S08848,244
F0000.S05148,555
F0000.S14649,985
F0000.S06751,557
F0000.S01052,723
F0000.S12353,857
F0000.S14855,368
F0000.S13355,660
F0000.S00957,807
F0000.S06357,822
F0000.S07058,214
F0000.S11958,253
F0000.S13459,637
F0000.S10659,761
F0000.S14559,802
F0000.S12660,558
F0000.S02360,646
F0000.S01360,901
F0000.S07161,073
F0000.S04761,426
F0000.S05761,596
F0000.S04961,833
F0000.S05661,860
F0000.S03662,055
F0000.S15062,277
F0000.S02062,282
F0000.S00863,450
F0000.S03163,461
F0000.S01964,254
F0000.S04364,831
F0000.S10764,957
F0000.S00466,394
F0000.S11866,458
F0000.S11767,221
F0000.S14967,397
F0000.S03567,985
F0000.S08968,094
F0000.S03068,340
F0000.S04568,985
F0000.S09969,734
F0000.S08170,101
F0000.S03270,224
F0000.S00170,408
F0000.S06670,764
F0000.S12570,811
F0000.S01870,847
F0000.S02470,960
F0000.S04871,047
F0000.S10271,204
F0000.S11471,808
F0000.S12972,071
F0000.S00372,258
F0000.S05572,311
F0000.S04673,436
F0000.S01573,443
F0000.S11173,596
F0000.S08473,959
F0000.S10473,995
F0000.S04174,379
F0000.S09675,484
F0000.S05275,669
F0000.S04476,528
F0000.S10376,627
F0000.S15276,893
F0000.S06477,154
F0000.S10077,578
F0000.S13877,685
F0000.S01677,918
F0000.S02877,951
F0000.S06178,444
F0000.S01279,047
F0000.S13579,448
F0000.S06879,936
F0000.S03980,159
F0000.S05980,296
F0000.S13780,531
F0000.S09381,076
F0000.S13681,111
F0000.S07781,143
F0000.S12881,293
F0000.S05481,511
F0000.S09581,911
F0000.S05882,347
F0000.S04082,610
F0000.S01782,860
F0000.S00684,184
F0000.S11684,633
F0000.S03484,697
F0000.S05084,947
F0000.S06285,891
F0000.S11086,011
F0000.S08786,106
F0000.S02187,073
F0000.S08687,422
F0000.S03788,996
F0000.S14789,152
F0000.S14389,553
F0000.S01189,714
F0000.S11390,243
F0000.S06091,660
F0000.S03392,219
F0000.S00792,456
F0000.S06592,752
F0000.S15193,087
F0000.S12495,077
F0000.S11295,252
F0000.S05396,077
F0000.S10596,659
F0000.S07297,955
F0000.S10898,236
F0000.S14099,157
F0000.S069101,327
F0000.S115102,672
F0000.S002104,050
F0000.S029105,715
F0000.S026105,824
F0000.S022106,048
F0000.S090106,226
F0000.S121106,573
F0000.S014107,601
F0000.S092107,894
F0000.S130109,533
F0000.S038110,979
F0000.S078113,499
F0000.S097114,168
F0000.S120114,351
F0000.S080115,389
F0000.S122126,021
F0000.S042127,534
F0000.S109128,658
F0000.S098130,377
F0000.S144132,519
F0000.S075134,382
F0000.S091141,493
F0000.S083149,286
F0000.S073153,316
F0000.S076157,890
F0000.S153159,185
F0000.S074162,314
F0000.S142162,430
F0000.S094166,914
F0000.S131174,703
F0000.S082176,840
F0000.S101186,164
F0000.S132226,948
F0000.S141229,989
 
 
 

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=0.1%(>=13170 reads)
ATotal reads13,183,66213,183,662
BTotal assigned reads13,170,44613,170,446
CAssigned reads in species with read count < MPC0905,105
DAssigned reads in samples with read count < 500354347
ETotal samples153153
FSamples with reads >= 500152152
GSamples with reads < 50011
HTotal assigned reads used for analysis (B-C-D)13,170,09212,264,994
IReads assigned to single species8,094,2327,484,905
JReads assigned to multiple species5,032,4654,780,089
KReads assigned to novel species43,3950
LTotal number of species1,317114
MNumber of single species47478
NNumber of multi-species25236
ONumber of novel species5900
PTotal unassigned reads13,21613,216
QChimeric reads237237
RReads without BLASTN hits499499
SOthers: short, low quality, singletons, etc.12,48012,480
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.
SPIDTaxonomyF0000.S001F0000.S002F0000.S003F0000.S004F0000.S005F0000.S006F0000.S007F0000.S008F0000.S009F0000.S010F0000.S011F0000.S012F0000.S013F0000.S014F0000.S015F0000.S016F0000.S017F0000.S018F0000.S019F0000.S020F0000.S021F0000.S022F0000.S023F0000.S024F0000.S025F0000.S026F0000.S027F0000.S028F0000.S029F0000.S030F0000.S031F0000.S032F0000.S033F0000.S034F0000.S035F0000.S036F0000.S037F0000.S038F0000.S039F0000.S040F0000.S041F0000.S042F0000.S043F0000.S044F0000.S045F0000.S046F0000.S047F0000.S048F0000.S049F0000.S050F0000.S051F0000.S052F0000.S053F0000.S054F0000.S055F0000.S056F0000.S057F0000.S058F0000.S059F0000.S060F0000.S061F0000.S062F0000.S063F0000.S064F0000.S065F0000.S066F0000.S067F0000.S068F0000.S069F0000.S070F0000.S071F0000.S072F0000.S073F0000.S074F0000.S075F0000.S076F0000.S077F0000.S078F0000.S079F0000.S080F0000.S081F0000.S082F0000.S083F0000.S084F0000.S085F0000.S086F0000.S087F0000.S088F0000.S089F0000.S090F0000.S091F0000.S092F0000.S093F0000.S094F0000.S095F0000.S096F0000.S097F0000.S098F0000.S099F0000.S100F0000.S101F0000.S102F0000.S103F0000.S104F0000.S105F0000.S106F0000.S107F0000.S108F0000.S109F0000.S110F0000.S111F0000.S112F0000.S113F0000.S114F0000.S115F0000.S116F0000.S117F0000.S118F0000.S119F0000.S120F0000.S121F0000.S122F0000.S123F0000.S124F0000.S125F0000.S126F0000.S127F0000.S128F0000.S129F0000.S130F0000.S131F0000.S132F0000.S133F0000.S134F0000.S135F0000.S136F0000.S137F0000.S138F0000.S139F0000.S140F0000.S141F0000.S142F0000.S143F0000.S144F0000.S145F0000.S146F0000.S147F0000.S148F0000.S149F0000.S150F0000.S151F0000.S152F0000.S153
SP1k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Veillonellaceae;g__Veillonella;s__parvula9417014501680249413441372560018875411840695211154125018526568970165152751213382311266271010579092465216743148312711427016880132320449610046861075769583443931324286768254010591805405295348132123265220302848762316107250190656712864231451067433782981260229015822265216612128110292179441361111285158010517315910565287552103295024137707181223143543743981470433518865067192014090886113426810122204323443520954090784378711491342681628953297757233269156814258
SP10k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__nucleatum77009260003351294230819504012790061150769226130017290271179851727318315114031839022247258342846780110608341471041211125191633786313935597112430146219905621915535146012820838894844508145472806090266320606095813582110506210155150388432980601261109142360741379371220215962879122119297010632830256168175820324014706283153179207914361601006014563050156752739174438623379278847702857262911188851578011188951452783213961600395246433168
SP103k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Neisseriales;f__Neisseriaceae;g__Neisseria;s__pharyngis0622872700000052396200120000043547000000019000003170003033928424021090515054006280003820233500228011307104460001200990000053184206510000020110000803612615025180004800052045409570968340350220972114031340000173910801280000000015335515515751753007970380000
SP104k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Prevotella;s__sp._HMT_31746635507660370325004802693069010036453939650605010723075883056726943336717703208111003063906447019617064101190154810200107233022617370220003017308443414016390180606105910306370096130218302931710015401550108133105781085013831045157439501783836073119884610812750319110259739928283521609621734175171007101094052815309511536919112619642778058106809994003223366294760579345
SP106k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Porphyromonas;s__pasteri36767824464498140950119198398110111274310299025011292400204306123452543321133119417574322049541223426186145198015542849381460971379912007102142227155592304497508474212641445957141972214704148266160206228949172593170135970227448632011803404631318904341962882616220712703691092341207995209145761634232404283031011345671563368018108454122329201392817332722121361005847016731968031420425710452171762014215779353361011
SP111k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__sp._HMT_21211540332155112938747332316151223451445450576320728507834973086925425717738091414204300101748795630106846030720431362144237364070246231656464234299360115022123012244767274148189551598073395907016303910120189224302834105285627576431457185910476601144004954325296902041492630423614034523176891890236337603679153441653552521768100293175613158982687390289640802211280399156150135720507306018221976309674672303016203900211000
SP112k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Porphyromonas;s__sp._HMT_278351158413101311917002081160200788024600102714526763461035276309414670642195035059119549130426001040651061313390219320213007500000681892411009202354094648502502201180108860005484610730300001471541132020412896901628151066191473504862666492821852282112275920821017148480504004320000027365765007409771952600000122
SP113k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Neisseriales;f__Neisseriaceae;g__Neisseria;s__mucosa00000381100613000002737682419770030015939440505773460601011005752212005332809495082061803420009447000280129339100000653901579186300000105000001981800002771843723270000000026000019920292715790185300000016270601075073800166850000000000127400199773108000174243009702690000001702953
SP114k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__sp._HMT_20300584430001557001392828606061513340010400046314354433352302001660835018400247620038191162041986002800001378002650002895001006200310002000080310219003600010400000184100023367166600437673140001980203012177295700042814818060450000278077564008330521930141002280081034300000058
SP115k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__hwasookii00062000490317369287039601690568015821000001391815802034330372401461141015800081100873523507504022180201742637008351055601062808157884090323402047900215700022453112305880028100442453000120116170208209030139191091611701309533165187137244021606311359661000541850000028102982118355108021915806860197841000506123057925101690000337
SP139k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Alloprevotella;s__sp._HMT_473014800001920363500120036000028512780069004377000450740025002859034401041903310100004800116003193821422206770018001302500603100178905945701070000991800189111025228326864502812580300706004012400001677021704282052398171000001410941509044024050300000815692342
SP14k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Prevotella;s__sp._HMT_4720061000043401320207927000000001501024405203340533510921802100015469374111790272254096028210015267300006800000036000330601100011313202040001815530037101050000144145473100221000040022017601202523005382835000011214270230662000020970042046000023193000
SP144k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Aerococcaceae;g__Abiotrophia;s__defectiva445126527043125493387898315501311320439756636636573324759847882871952832812585879120202995216396235667112841110323662275461441791581872692362155155913451280341082727533199453001553589583625127191748211511203261433467375113246480203428162428634696254427523109116932841145556685463893652011411671532786196539529074648367595014084188521521483218471646381330125417023131967580675672732347110272194427403205842890121601574132484243426245172310628213009746664276550
SP147k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Prevotella;s__melaninogenica036121123161317190228525314014018913718760019391531714480312768632250459587697883443339211104818926023614007665751060011113133051875429770235445726521174230372021723542100395236142429513753302494813273423092039546415316528838943829228153003212693019428014316480363191602614322238362413354800322076451789821499264658101157237468207720357200613767389990171719361427
SP150k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__hofstadii416673404503020000316107215226162106065010227854008867030557128032201163198284330170126409000434362607551480000987201681183700099493500000166943750271760118912005030302404001510320003095047072038739611598576629865185000016191112001375534407892407024818951210809250276640204055900108481400000841173003420202014650677391556147248503997
SP154k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Lachnoanaerobaculum;s__saburreum037104451341012700268909397000022575497000113112260017522090391490146885301991400000565875413300000300330250000306141000492710213093027808720134000862163016000178000267152509003255487132300244187116003604732706305273431172710928360891070056380275010708220404602121752010906510219001160330011311587491670415301
SP17k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae_[XIV];g__Lachnospiraceae_[G-3];s__bacterium_HMT_1000073210147555413121159838805403262811984589104161533250331191572054224176906531091201304615646871018962453276259762762101450151489014110489154929807850538596094000291256181694450548026040370798605850687012781166211601943217052205157375026035612119766948384001220380222208360433683110186152070040215930404282962030459407344
SP174k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__intermedius021223080000296201930028900310368018038410102480025110324342800011900461531860085192670011001610400007518121368015402200081085821588825243026828278697191067257121292500158761531222121181127237700012156601726273504322334511094280383130190184427012461742462340234122158191230345923818727313320942111817726347040680180008826972094
SP181k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pasteurellales;f__Pasteurellaceae;g__Aggregatibacter;s__sp._HMT_5130067029230078581326590525800018950018065837227001050020576128150008819813490000132739567386982680828510369002300154620002530041455052881065645790818346665000006607270034003099100403580264902300724602044222406971113604052440291908710002393465105201836007910684211086101003000000001185
SP189k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Actinomycetaceae;g__Actinomyces;s__sp._Oral_Taxon_1713220061005576300038000536295000017900001887021755000107030002430003780000009566370012202801740206187101167077761000008900137179615200973780656001170050001967692130089012301740152000025900430105000000277010501980173000006400058000007690168100000000528
SP191k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__periodonticum1281092424921233019341361770039209691002020154295541466865035016145793000365309480594311217296026492061863525700537050323469181139810130452146076211072598592819326284302100342528804863626354013417121447058456792331170774131802072831812221131005006125820890982112101929356112845188085206793360140366220107115125591900711803018664919712023410900660124891
SP192k__Bacteria;p__Fusobacteria;c__Fusobacteria;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__shahii0731603200699906996387018322969310425105301170953889831162005001018099401925124600354400793839800263127451280701056872935349414898191757781321086235233027732160900139610381313427152713172073502285492188810445699416700193300795231130431841181079655061283155412803039010216619100327156537874712241335568363480105390144871731009653529416909116617544492448878023986275537911517288400564733052221370201662865662471011467002012457305020017755485319128313001047159210597000233
SP21k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__sp._HMT_417510534270169330000216016238061295206593319584670231700527002994362803149108707137045171720284010755217130649339001000000014337761701801006012372075334364245278507839063777113700393806021460017101276721901804704174016174553240579054490153111117010934694263506051030307040069000471677724331900018550563384100015762336302060200321
SP217k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Staphylococcaceae;g__Gemella;s__morbillorum07481840000452741793662201130364253472694386171509414093066975309108161425023805782448142211103565083087901224301169479838085026000477238512102731940390151041000148061585034072700016419780571034312202088331525681767377017304380564308022601021994871002271680159290367624503131730128304374902304146179069482505382263080015491683108500609345
SP225k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Actinomycetaceae;g__Actinomyces;s__naeslundii161043833029642346457760724497199164214180410820884033915266913392108325641378316082732662560203284343114123311488013318010069023710332514438120117610202804333246010444134772871336212410611645937032332974924526603662994175695312217613134431040282111026551374034206828577718121800652138113924526584980239427312983029201761201687248596844556451501296475051272221292841109152667721562128526261929919461999411521361314960584824145819566191421712570160560154922581314
SP226k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pasteurellales;f__Pasteurellaceae;g__Aggregatibacter;s__sp._HMT_458540591343920004601134982168002301302872841511366459573407843833012919475073743102494685330762806470355103056033924396103313891864001613000376460370211580101189716817106710103329132020212813800073277002471250073239354423301055001085771010232032181371549269300284126751114750133001718840251103637397341433013760102339433208119431125768600368190548222
SP227k__Bacteria;p__Bacteroidetes;c__Flavobacteriia;o__Flavobacteriales;f__Flavobacteriaceae;g__Bergeyella;s__sp._HMT_32219523129713046335874363977521596336995055421038936147733241873306765526763401401756878252111312037871952662479326526287710232160212107813761613197345101313653262373866143621831797165507381213279872750291644381716660979109610092875419423211189203744053107013182492333424612420603387723753185569835263672695891016654189093195331117325354678069465158041115483462824239520488632572768111223573403
SP235k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pasteurellales;f__Pasteurellaceae;g__Aggregatibacter;s__sp._HMT_898060140000554170020081165500100107012000320106001590301400004040452022820297135512536962002060182007001242126700025001130196025070140000272090057301242300414322000172001182002316095416601395400032217284001881329800013152062151009269191222002704813034024201914497200001550100
SP237k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Corynebacteriales;f__Corynebacteriaceae;g__Corynebacterium;s__matruchotii110941985775310622137612134056514492159915510132532257610516757220464531155991027715135156333230160033691214448234106053811310196234888395116739759271574967411515589169013987944280230953766298977314443372425735262287742291033652174567010500154878833599747712297842613093292685994607570332208103391193028503172559826715411455714776112554137449225723147115087240101488981231410213375332723501640001347173237157111097123158485052197656259935651457678425491703822631383553411203361318923854305520539142710219892676231870170819489446387182961496172343173222107643046675119019949113555930480365422063336286281150849439873411743512812
SP241k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Actinomycetaceae;g__Actinomyces;s__gerencseriae04900003610003112091801442000013300000002020066071003506470003410019000848157005295631005011004500001801019000000200000010200019059014001500020630404500020162513100220376900510000004144801014144360126500011242000000000021
SP244k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Carnobacteriaceae;g__Granulicatella;s__elegans81152181497542439728011045290004817711900641202323418023161018000423397411461395770150441070903245019071701307244510225414151515442926142980011810642701254648186501602411008020006314291916326586395890131242518431329220151452690016201301491951613913801090196275680003591292948125317591865341595000789582
SP253k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__sp._HMT_392000470022022988308464831000474064876502271732671005320119765215102757013431088740013137335406266410164815203150171584200241586185041005465900082390062011503516001930203096005380002875702226021359071315570244304742392740250162208046910601645051107621500166111863128126002023213022111752970142452713300015418638128929703772761242623463200000756
SP257k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pasteurellales;f__Pasteurellaceae;g__Aggregatibacter;s__aphrophilus001953340006000000368000047029200790093834600034271000000111389076600980057102260000000280001419417403016000690200000247000133000000141000138135500000760135870019063500086721970501520141336000150011731913900863850038613000285001720000009645500136
SP258k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Prevotella;s__nigrescens00800021022901090149300003690282042604206510017542002430067688331000285418101371024070210019832920001600650045000200137022258401409000045005073950009970000110152660161404189754303178000280115003771061420319226158000035409263396035000204125005591002606000400120000
SP290k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pseudomonadales;f__Moraxellaceae;g__Moraxella;s__sp._Oral_Taxon_B07001000112019200000047000006007007870150000032000080000009383143300900000000005133500000053600000000000000020000000000000143816900086058761400022130000005200000004857000010000000210003000000000
SP291k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Actinomycetaceae;g__Actinomyces;s__massiliensis144155495252590189140510950992402353839143681634224871524241518017090105131983162656385101313212834321022891691575263801633766217111110103279450137008414313491115251168088072694684511131103820097815161065123519443101952115075523666835379618264613717667285611113330641012534245183612108908132110440331162992634330610781131487252719035428216981097038401
SP295k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Cardiobacteriales;f__Cardiobacteriaceae;g__Cardiobacterium;s__valvarum124018370360452201301210000115318621840318618728391090268817815322871119114000128021298070742713945348381000381267120015456725025352058334703404093234201303242900017570000150230045290234401816426575459480591927706520571415806517112053002015903042312343101485416312002531403257400052800494
SP297k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__wadei116200913002400399200003500126378000000411389000461584869000660490000171611741000000033304215901773454700001945181880040190038600152265223545680005850000856215900978043604331520082573000021080168552724792000506500120000001980002459487041327344800002104100009381724211120400242
SP308k__Bacteria;p__Fusobacteria;c__Fusobacteria;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__goodfellowii001482100000012100018253140000300003570320561136020108000000145320161101033280780533259023210152067601985016800408000042000001522140000111000002460010565800000366320031055203007001200011900003000012000121074000167800059412502416012000016421001742
SP318k__Bacteria;p__Saccharibacteria_(TM7);c__Saccharibacteria_(TM7)_[C-1];o__Saccharibacteria_(TM7)_[O-1];f__Saccharibacteria_(TM7)_[F-1];g__Saccharibacteria_(TM7)_[G-1];s__bacterium_HMT_3464940000005900043300009731848312400089013001320068900212367000001390101011520000305000007201600000200374442013700490000082000089000000000690460003037397037701001305850152801151491260219592402800003606821601562332180007750000009000000000
SP321k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Actinomycetaceae;g__Actinomyces;s__viscosus011690709514613000000030420450097000339901091190000004830602846925711203111690026014220911501130015640232000563016039338291250402400348846149440167288483324920001061960000001962380946800274000000570206600287507100402126101701210397026024380542225251261600203926019000122625294030800009947725300189
SP33k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae_[XIV];g__Lachnoanaerobaculum;s__umeaense7045624817508259666162746801716992226658587316141740576026651670212146366225317158062984992201901042831465985008570426549216623467932139219226413426058471167611793493527581472245353035580800821483239140686100003430165201181135104714927532027128102049121613382637541894845390175651373345885212706192423066475724504765380532209727310119615112238141074242484134441334412416846552225603044818135296
SP39k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Porphyromonas;s__catoniae0003330001511100000000220010502771210091500000983089000004170130008324720000000000000000704400138000022191380000028000000002200037014397000002866683058602300160672000830001965000001690017008001895000841213550315150000000
SP392k__Bacteria;p__Bacteroidetes;c__Flavobacteriia;o__Flavobacteriales;f__Flavobacteriaceae;g__Capnocytophaga;s__gingivalis4607444002250162346529006118434800826279656261410580425135530261958671017321503016232302514520638380021201521101091762194574817003340614800385919410501490586321241092573482222433326210504592007241290660640308114931382012403534261843715638686011136428446209239926309232069301412321511346470133170007126202288213427000254600185474
SP42k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Neisseriales;f__Neisseriaceae;g__Neisseria;s__elongata9266222594316785242531513154010662549030212874109146710159856330228571671500021626944769040883310421714219270960228179585178513426531054252672162549069148230331193270460031619044633429598149937702881188207104300313056514130035116840771966111022581912282146025447829052575688415183302095256814333871771654330884140203127424713516512010271405142253534902052130001699257192
SP420k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__hongkongensis77823405343848341504309035036187300013227523815784464122384740437210035983360951109130288112305144835501431705445002763532820116014041021150321327719106171461471132723227102921393501064016292734116401246264119435236632166430949615011972171608170462952057143301337097291034352582553065130979225411957722017981841636383188826820373131141330179196713000747440401335682159430780121745533768400361145533114055031302641754571811084411510683
SP427k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Micrococcales;f__Micrococcaceae;g__Rothia;s__dentocariosa534343049811501002000241541044225191945030962029922711399414003921041207003607941512529417371707176131373543000085005390014110675570006586535312263216229327332443751415280272217906476548834337636416280316434220274900701118921074739594805432278731682404711127013704435580411735148142130932332200505069010056274472706637242433201830420409825058194814919279400005741005448390896003550827403393141925
SP43k__Bacteria;p__Bacteroidetes;c__Flavobacteriia;o__Flavobacteriales;f__Flavobacteriaceae;g__Capnocytophaga;s__sputigena4203459132930818739969336921721403319402854102554160201386215441716674417892117126884201182071379533121649131229051882761044813801965138848270603661179422751429497378661015588357247072827716174394058651047001644277481519002684859907395330516701209241111011317923217633154583589107418812512244196417271281363403884283737988469535291226805317564252741031232344614022836912666124587112762377842143001827475364263
SP434k__Bacteria;p__Proteobacteria;c__Epsilonproteobacteria;o__Campylobacterales;f__Campylobacteraceae;g__Campylobacter;s__gracilis71437189985890255220169721282145716731651438886121193212922246310049060974791802587284116054930820103616142245182914818143432591910819702736420841636902113085621781110135003007945177163121369719002879115748529421312340254150664163843052361046933513239973178151128526223596327056548661980291164074754232110026941436865422212013008738337138464779727314792345557399131157248931090313288211729467268058
SP45k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Actinomycetaceae;g__Actinomyces;s__sp._HMT_448000000300000000000300013400000000003000000000029800019000000000020010018290000000240002120000000000021810000010300000000000702601000000000000001302920279425780100001031930000000011
SP450k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Burkholderiaceae;g__Lautropia;s__mirabilis9921271752137934810235669218101455373633570102821606093134110383837826785150839120317140131930202718298238585034040192858637621113131920492571533323214218363384831401250745373702333444718435146075482312361330965184422042821712499111523216531739190842360367886250114994333405724612115926516136675583415068398925232945852784441385209862187027159496625113729150362488675157814882452621519101854084963710011419231845033301476142105610675119416366574647261163110184865983708494
SP47k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pasteurellales;f__Pasteurellaceae;g__Haemophilus;s__parainfluenzae4131323387313422961136562106347621361848029371384484016081163452665453064552481317956212438402010291548013744528945520892491050726848132857476139538688114222853714766815017541241430657540027885928143116731864811152961982703377730822156637243298108837013975415876769652139530081262135714814757919684334146261062912663466398900363257935231394317986045100849462823347374354610427663759229485812415893093103411280741012814814090135983940750952618491408697277136662817657129389614941591118147723071663162512212045271941754128
SP48k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__sp._HMT_2155315201070003700000098049204121220013015600002501490090000485100742300006840002537094113501750695461000001673943178043923632937000601420522715750060880501900131482025802079489003030034100093016734820033623774533018026800507691224301202712008502876002499810527290327064928274
SP485k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Corynebacteriales;f__Corynebacteriaceae;g__Corynebacterium;s__durum1985240341617117633703177474286432014017426562632218968585273763327740155077274115770031017231196806412401057292392884433166532082310366076216935817114318632551718938382335611296402314163230921117498782257132846682573721221057296838868793813120112333923162370118711873132313281345724806799315515254632629522455031312163962924182980114130057831872661357512232872512005027937031150648133117592028917138167917701865041052891224024276912216710571394470865594432494037414874198332707565188771704936
SP505k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Cardiobacteriales;f__Cardiobacteriaceae;g__Cardiobacterium;s__hominis198180766342310550227166859410013220464371725770852153724931425028191182326930632364469424328307843259141412663173318522392496651785852056362456640711766795993233678496490716805931011047361071841349238140237202583216028779031021710158303176625112344153513304102858264522022868903770841585653471073227302092611002914723613301592810010354021263255499107761120167309213942795771913001182498133788
SP52k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__sp._HMT_2211700014004048112400030311000392280000032000000000350530100522510000022160007601220728267152040387029505000023005012305070000000220500144821015360003400028160104003228001600084180033040202205031061122001200229759025001100220794353
SP53k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Porphyromonas;s__sp._HMT_275023013500019861571703000000631111522245600015604631732843062510100187570016950015109727590000503459475024761010000131000886008261085600120082586115159020706662700417288700132676001328038700470017700870380017749923791202220000001100530000062213300064934171300000001450044158
SP530k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Actinomycetaceae;g__Peptidiphaga;s__sp._HMT_1830294192701330020400097300805230239000112300000360910000001646075499000049899480004902402238085000000600300601700110021000025943163990030000042000962065150017320098300129007300000115003329000005050049616654909160000309003830194906210333800077612
SP543k__Bacteria;p__Bacteroidetes;c__Flavobacteriia;o__Flavobacteriales;f__Flavobacteriaceae;g__Capnocytophaga;s__sp._HMT_326001702099005506237840820801295641722601649013300541728331090569403167801205722338953929178102011105026176013029900723707301080000006601500001444816100461110611000097066810006211491822914925335258183657005163374212035113562822440472001241015900021901353109001339021296260019025117740194890087129
SP570k__Bacteria;p__Saccharibacteria_(TM7);c__Saccharibacteria_(TM7)_[C-1];o__Saccharibacteria_(TM7)_[O-1];f__Saccharibacteria_(TM7)_[F-1];g__Saccharibacteria_(TM7)_[G-1];s__bacterium_HMT_348154529840002574633987648465114410850420037323365696063361314360097013323436507101383637212647900162043013892300477231742505170041325001100488112142926175168803320331530410186773660000487150011910540464197651622053630688310493602982419201765212481127245147540585991161586940104611700000106123938151551674170072821013114743151396800077105158081387
SP6k__Bacteria;p__Bacteroidetes;c__Flavobacteriia;o__Flavobacteriales;f__Flavobacteriaceae;g__Capnocytophaga;s__leadbetteri001041030304981171101854286011419295057214019917325118621333173138398802488074248213135146110082337017517231944209312881143222583141811911823214192775049311620319133353020882948018938133034091377165497122080162202128702340656371009929894865811393216344426809110599200162821222212584261271796190191182421038502129681101364194191320597737430020531613514874744729532502672350193339
SP600k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Micrococcaceae;g__Rothia;s__aeria410284977163622953360865053322153554238233882912404925601140553010600220250840765241650007767627315412742863730183803321602688020272025730241664514022467750870382478667799014214528262471763249166995955168960301014187756887876923019824214128323222833582054649211937192053520172461064633014983624916222668210412263194179303835429117877131304191486861120865981153592793263402716102387577605545180092922765701814
SP62k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__buccalis2185037460800058605751302900790378310951566780687500189349205006577979442717081238685031528100242004946438536359549157443844506916666029007319400206138034478326408897094427608860002365085808700870007001022029920025980160500013305813104503291303010846981040249747625823677882217175684326103470185601400639108001281251701930650210190021825282911470247642659000061366
SP622k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Neisseriales;f__Neisseriaceae;g__Morococcus;s__cerebrosus000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000038000001382018147000000044200000596400034500000000000000000000000000000000
SP63k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__sp._HMT_2251020229242562575493669210010610050301464093269478009305540621644564664395014313791624415857836130871800180846600013462357119470102722215516289568392740171186443606817680255024114690601368921284014018602496451826150495049279323466060111525059793920283303410366359746221938310571253758049531307589609071236109328214931692458190700212595150213284506134636816192598310019663839545181606372189423387440000004933
SP64k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Micrococcaceae;g__Rothia;s__mucilaginosa075141082740008724089661610189232429011001616157026014000863395203724140310700060104920251401234402411163514660036399876584160212303681885131900014536203121560304269413813694338513208233894308823826065731062640249514522001905366581316020103105700002902297028100812502139270956
SP66k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Leptotrichiaceae;g__Leptotrichia;s__sp._HMT_4981961802000453210445180481226510006944280430600033022000125100304800015442822000001407370725800000000000599200000160182010136001700091053400021177700038118000026738026528441016649018760051570060369003957575000640800000008307641540000000024111000003676000680
SP67k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Tannerella;s__sp._HMT_286130572600654022168541771070242387362431274791349193417808918971361155088013727016028491673192498371542318611305879617621415004023229070333411010173206470014851404381080642026024320600041028640505891933171047125284333853332409002991822551159600149700025072010616828122313213208141719425116296303381118123275044600168
SP72k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Prevotella;s__oulorum00025805000350102400000338143821106800301097210016188595000871550000005216761613002401517306000033002840193091808214309000129000001513203401500000015000950283072500891241155690321563500120103343264000101244020179060014191011068612602182600203910870545237055229090022
SP74k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Veillonellaceae;g__Selenomonas;s__noxia40619447244820388583324058017630069039834015170451019019936659019205610106324317614469493730040585490257744044309326336596107847260596775511317972837600445818886051477843352711900277396110853915311641054900031643927700230420157281718015685059121902815513046963323937104013111019845213877013114653844326870510418102731003241060459290151127101301233331010303616864994954980166074182
SP78k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Neisseriales;f__Neisseriaceae;g__Kingella;s__denitrificans9316130548101304730271868322908060210443102164166804169220215828277117423835009353914225310010627502780182332003106286675319044628693201270119899198988130108510199059246315371298544206193288106483814156381021007399001162324079404910621220171645952359441246184858582725561232117617347913648774431510035415111815224411061264049793711547600124100568665
SP801k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__mutans02100008700766000000320000000000000000000013000000000000002901910000000027258460664008236637067013013401136782000400117400000004122000013106002917614006021000019000428000030386349316510912630000013712660277070794136000763
SP82k__Bacteria;p__Proteobacteria;c__Epsilonproteobacteria;o__Campylobacterales;f__Campylobacteraceae;g__Campylobacter;s__concisus412064131481803206913319631012369280106519105156270325115281919915231129438255245328875185003827166041778490514715190251681940156213713521014235864923702193114255753001503144570802073580370002036166174137204025680180516828122124308600595633059106709475182751721116333081981264346601580168033510140180300123201061900622639412015114226115773521073207144
SP83k__Bacteria;p__Bacteroidetes;c__Flavobacteriia;o__Flavobacteriales;f__Flavobacteriaceae;g__Capnocytophaga;s__granulosa02296013014019802625053886420005997046730585220840541251251130244303260141428301139565357280023292596135955027014311039247788992227115465000002690982301388203600224053081669611227549441030472000122630285202973308451336464244141764341776042375965721562381306811635179301320290799573013490610025991610153546942560711933902291735357042185701320151680178680
SP87k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Prevotella;s__oris1401730000211004770459000474102650790011014121500000487001740000130064170247022720490000000002441021783009036494262726016214700095390005147031004989129121000043275003081990941210109403801153309210000278822027901790260014272731502082013808768060000440000010311691000
SP89k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Neisseriales;f__Neisseriaceae;g__Neisseria;s__subflava032178346000000122913716400110000025008700005900220000068400017241834012642701320078005562000812301932002809003455300018003100000224532543324116100008630000447055851170220000047400010500599299018850130801076800190000631001500000000006618000037000000
SP90k__Bacteria;p__Saccharibacteria_(TM7);c__Saccharibacteria_(TM7)_[C-1];o__Saccharibacteria_(TM7)_[O-1];f__Saccharibacteria_(TM7)_[F-1];g__Saccharibacteria_(TM7)_[G-1];s__bacterium_HMT_347137075790026000079592284000829000001221009400163054210095982409700000025807703300013403605990026681409000675070790250057931214000019720344012060000019033000046458001050003460630412548160079206017200052020227700200715807692185228197009100001983000159964707
SP97k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Neisseriales;f__Neisseriaceae;g__Kingella;s__oralis543218451334350386121011894577402487922534052791319508936922203948218074413012115117565103173642287511333016198704911415581140885163157125306682483127885184048811550018650949641750816110601488766994018463315354212601438253902453018715842430679964879315617811923823860287639852145573156193636758249514928214342160511403071541190214122912006491611916931630426602512982344307936117156218175653731913614566181617772961170186
SPP106k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__multispecies_spp106_21508463376171867403604135064491511025086732693516115012770776611791611163151824666407154536191441475449467356426142123112262439015921552797351646538445223292197499125980863182040176870481423456934920110475649682169910432256758032445026478663045117611027207218130732741413436911264196952451014559473883981771114034844901371385158432568827104393982293465851093122573476395160234376742179273205767921794128404119368787574167644962593041304673311484375858119057362112529147391164228923179682234426614521646298139343534845044876038181818273101913480421292201343473119361103343
SPP108k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__multispecies_spp108_212004447005104130575502664400018901715208250067320006040023005810000780128033906700035212793083190011149000004320135019730027441280106003510574290000006721177161001550552623990081513000400023930244983560018171065892739890000070183639388753543344601123101750000941040052540395
SPP110k__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Rhizobiales;f__Phyllobacteriaceae;g__Phyllobacterium;s__multispecies_spp110_2020149000150007006607000064162500740074016000000064713912000800000000001080750129000456029800000422804005120000000000350022171163060106327000057510308034900000900002495501700000140255876061013190120100027522110711434660000052011007
SPP114k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Staphylococcaceae;g__Gemella;s__multispecies_spp114_2294380552381818197363227365454761191971213207015262582518152828132604491626247185394231601417229156187151711352364738968648023357152572376557111221329952020617556943632144957459820571662532138139051033242942270381504301845518167150147242154135315290201773571355033903505711094611444453435372865173341229138183800114231704127242781497171928513359142361330164087128864811274036128409628392855
SPP117k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Neisseriales;f__Neisseriaceae;g__Neisseria;s__multispecies_spp117_26516550192133037224754685000491385087137610262801089311020422190193301826268007116060353543890021028200881339030235983002090800299103010551705010213003566018290000820001136257001782233134009385406006663854811311401008809278219314395376331011371902301427413059129672115466152830600815103011810237873452000388482143
SPP118k__Bacteria;p__Proteobacteria;c__Epsilonproteobacteria;o__Campylobacterales;f__Campylobacteraceae;g__Campylobacter;s__multispecies_spp118_2110861801941569459551623109021304191559087488514838196157271333391766358004141445791931030069232333618141351157478509617261645133947181335343171100373720210844695024336609308061041014217569274000201150631731274011915342805231175380101921681510164142042612274941629235043210121213603481350234729515121340660131010822435248454043908310692949175185017160
SPP119k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Carnobacteriaceae;g__Granulicatella;s__multispecies_spp119_2206648633914336291303210211810362062243019675111794613032877643132050246129354761216914531893091643809445272032566118127913692588221027157327427740848220590863411726850324627186013657231573133472481641043855560387551222147133202612165411623661421897761240113553879476028562323259264456002429103778437324607035695682311821885157470824801673303321239416474157220252237974464914461155557680524741186187507037349190913016794885339573833418158413026323036922139501425286116920218906691790
SPP131k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pasteurellales;f__Pasteurellaceae;g__Aggregatibacter;s__multispecies_spp131_2011100021026351153660007041206029943530512461233310001093640493816939537287318980519002500001730015000752400023619040010232300000000364023914000660000000151260143225938380017260408210871102200560240822031233900900035501518308034000080000600290015003520311
SPP14k__Bacteria;p__Fusobacteria;c__Fusobacteria;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__multispecies_spp14_500665000284570213361000210274212300011250153910320523000011300275059000000456441073507100340013400073007003818017025410189420120000122350761016008602700055100000312320216329401860004790410018411501410193900067506309734520159000022405152001780000052036607721
SPP143k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__multispecies_spp143_400450000000040019000000003100000334000000001841800000000000000089000950000141662970000030196809670005843027266000100013300034017292600140019000058200000130240050000800000011424496860384899000052750702140001710000515
SPP15k__Bacteria;p__Fusobacteria;c__Fusobacteria;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__multispecies_spp15_500202000323190140993801821661401716324161006180125844287230043200104670677034000067010896018242320529831936128736703680193040001702214602905977918397261015600064665501951201630640112110901079515621190152029227439439680217731602101620279049104229069889366183001885807206981120501414803767569059612006004284101917910807782
SPP152k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Porphyromonas;s__multispecies_spp152_200780001716722106901207063836200004666087803221242187819071766007151220179733210254551630112012045532791337387516529943025138746481122049429900023519401953410193339027501900000832852896980416713233703050750179410911124433016241200134800193701492353800236028000077105755700000968800642800150151208800000145220235
SPP153k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Porphyromonas;s__multispecies_spp153_349509033801052624065200001447003450000015539769015972085278582101359777680001631210002224414100010827700046000093777110112410596600002090360018540000003112780000147001167016267203180077000490086205108033121833581205024160181402990001500000000118120743101800000000385
SPP154k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Porphyromonas;s__multispecies_spp154_201920359900081580002382367000892007040602012967710400875130296510001080350021900048820347181538140211022907302600002622207000000420002800004571604780000008101310001327569800000799184521876635700035060204030327182341720242470090003257991027029320011121213700049025400000001068131
SPP155k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Actinomycetaceae;g__Actinomyces;s__multispecies_spp155_2719891944253034437033001310176403700010100344004000005142200510440492150602011073111360051140228001110105509040746193525034468054138153575011710199107978424108151973608015901013216902260024218412791871901791651976016011789502402970144030018720484328615925908806940058205910240047120000144202191830451007000023842479138026060312187044974363209383
SPP16k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__multispecies_spp16_20540230055634416410572301605402497270180000167000014970570776200382093000451035260099816852300010762034015050001331870183054300530001910007500003339015748000215001040162121506900000054538084192002313070251008026560098424107003000000327016000028936405685018160179502011500029489068148
SPP185k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Neisseriales;f__Neisseriaceae;g__Neisseria;s__multispecies_spp185_243181004108773028440230130081712440650204447132002002680002260000005201893887467620000928900040133919040143160400652240040274236400034150300103719201382320433411300553899901626126026810049943762120188211130328001131690023132812206040030401111074000030004562
SPP204k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Burkholderiaceae;g__Ralstonia;s__multispecies_spp204_200000070000000000000000000000000000000000000000000000030000000000340647000000154230089000000000300010000000070001700009013880000000000260000000003891107585002800000297320005391900000003373
SPP23k__Bacteria;p__Fusobacteria;c__Fusobacteria;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__multispecies_spp23_3000800036300399081001909544133000001848951020071017460236391950021302063705062000004210840031536401810084000022700000432370001754585435013087000107000141012005237136046663116601844298513233501280048546025174030000009560001417932610172036862354440059003200252000061
SPP25k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__multispecies_spp25_422024521181134175720265304521629672526992256204025352640673252348788738515544272467149515884071681111715112394801565087542737896362333401864223422084300189717844261402306331160102333221115149135234119170081517324594486827101942201876661967925422486600396152612210081720998418802104222827112550241629325462634410909635292629919776312764226543915614634551422299899109858073769116111549936211258839054023269399059495583124591410064516145246810090634837835131515551102138811581936814859121056622219349928117968788495393534991148980235210299926354066
SPP252k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__multifamily;g__multigenus;s__multispecies_spp252_300116280045758637321009290217816163722587355101801938032882609502017336165800159011600054436278003165831510321017900172005052060000001734340470000106989706443200016015509146904005902830011811615356081213138000222931104300081243701999286709911313805605095900008900301025600036032500
SPP27k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Neisseriales;f__Neisseriaceae;g__Neisseria;s__multispecies_spp27_541517126726828841038313421654192521768171145803117371957196711882473303479613417808416644385280334413952795107516721219200673996310442981033669118810344054794376686787312833280172274941741791697801967041534231684478361814118118408721524222066145970562364969401206270343321753554372177312824415367326956454423678828135511301732502978013163664376374322218104743409051762423317471997166013851383368220295421049510461809662671087427692171025518331019008687349171947770247307261931140391227087491519237244611303345110367601087236134803
SPP28k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__multispecies_spp28_300028000276100970502028044100660000737515301063950065019340281000295006224712510362627306840002993100000473169045043004640081000280250145005000010200000376000652012137570027477080009506110633645760006097360000000116601413570002533640322006700168743600000099
SPP36k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__multispecies_spp36_23929672238346192108442190066510831288502983413467052841279342491222374472319440944691521487334429108767615501391286369681456181856206108918220001316551028212015669829102200128200285227477183129462139160616821641512123755050095732254887913689291366100381222250922711451538281763562436513350812498443123820642436995176132111212132606408381517383182314847361293158427928327147804440662180515441583131249242452876689437523066925941089472037637623496894734461135
SPP37k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__multispecies_spp37_191307723995242865362550101591112927342976283157917893367599530492432186334154624305490731748841853125142924335072459486839191008198710116411618061663224922393306296169381913544433536620372082257436253207154551069303521911611015702635891388250411531320462157821631041636690314272117129265597593933760888955469523637689351351519701006162252296036184430740865179079384795012515233594019988523110655711364912883320272472190010519388526034330725221073151033085815761203945186947346901728684046191019163292255249439140156589018440482662513265037412735627491843397041269356710811207996314803366705971066236291951065157711211132036621067186801911619387
SPP38k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__multispecies_spp38_200000000530000000000001710000000720000130170000008109100000001641750053000000001180000000000000000003700000003210000010301770000000000120200000000069300158000000013311401214186000000505000000000696531606
SPP56k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__multifamily;g__Lachnoanaerobaculum;s__saburreum65007340052400125687283004301835450007200166455278001754555827400038411120501300280029000000111446039600321021839019501630436003036000158012027240183212036901300001410034505658714702582286150005160211002796781220084372008700137007201710082012640000609351039001041630000236030800116322
SPP68k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pasteurellales;f__Pasteurellaceae;g__Haemophilus;s__multispecies_spp68_214381107400481640000303845000054250275002601802293003965600023475900000702515230619000125673902602141301045000183709300814690015901282116344923790000110260926288031304110797475883905806431013106664710015005014212116170001414591037140001220341303612509000002710602976
SPP8k__Bacteria;p__Fusobacteria;c__Fusobacteria;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__multispecies_spp8_2001250001030076384243001020190024200104304077916009385000551065000000368249450637115180000027005410002030014043300000000000000032190267420001880310053652000000000196002122000376000089121001092700002660400154217028100057931074148381638001730034621340264441600084
SPP80k__Bacteria;p__Firmicutes;c__multiclass;o__Veillonellales;f__Veillonellaceae;g__Veillonella;s__multispecies_spp80_213501263135256263512154160371374848186617954714641318527508437336816739105258683717534964768426430593741217529903826776796153132378159929338769671658122339328354410151717793547368270211550041316445662149040403531114539362155112811667167413006009714117155826406132682753277593710830881180815891764640418633651158521141124229327256494910139021205360304142561223882862233219106248066631032185101767910513056571911360115103446989630893552411537138617449573429131476517299029139209528161866270031426112581693
SPP81k__Bacteria;p__Firmicutes;c__multiclass;o__Clostridiales;f__Veillonellaceae;g__Veillonella;s__multispecies_spp81_3001021324027490435312414916176901509084812033244642003570400001861329244019853020105141815016112016260381361265113047033372651004801811821373033321900403740736378151100900605695664043553851104501026580012746503643091392571917172814348795163496023013224332536731277350021304842282826771261504517677136
SPP82k__Bacteria;p__Firmicutes;c__multiclass;o__Clostridiales;f__Veillonellaceae;g__Veillonella;s__multispecies_spp82_300011890027700479000018500008100000000441426800023928900000005000969126017022034780000066634330000000740011017200060106600310200800240833000188500000000000231007580300191531910043220730093805199086007074008600000000065001000
SPP83k__Bacteria;p__Firmicutes;c__multiclass;o__Clostridiales;f__Veillonellaceae;g__Veillonella;s__multispecies_spp83_200131149702221390804351861542991132122600560194376310031304930043105800314023102836001589502523013138103188142255031130195373041044534003800415120637729041201355684002730134252005015420010700115712920003652397850563025064002908501135310310375428529444516004621090944703070775812335132793779660016405838148217152670047420136195135196
SPP9k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__multispecies_spp9_30001500000000000000168000048121000000152360930400029760045731921010000000000000110000000019905440141000025004714400000000348339000001500331100000082201230010500059600000008400870723900118785651280967100320000001660000209
SPP90k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Comamonadaceae;g__Variovorax;s__multispecies_spp90_285360122752888000015063011271090001412105001670039211010055002806400121076871323191127000000019170156620090715177000000000000000000000000000000000000000000000000000000000000000000000000000000000054000000194501098340
SPP94k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Staphylococcaceae;g__Gemella;s__multispecies_spp94_2209035021102144105435170002467963720151031681590409716011622623221646079537622524600579576249866044041816965500412080514257219944932709138426103123464201301040649018911172350097028116900741472430140431036132369081860769232102227521967722429702723783978236113297745022803021650101147739861801201806306314235471673019300000301150122
 
 
Download Read Count Tables at Different Taxonomy Levels
domain
phylum
class
order
family
genus
species
;
 
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 1all: Cavity vs NoCavityPDFSVGPDFSVGPDFSVG
 
 

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 1all: Cavity vs NoCavityView in PDFView in SVG
 
 

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 1all: Cavity vs NoCavityPDFSVGPDFSVGPDFSVGPDFSVG
 
 
 
 
 

Interactive 3D PCoA Plots - Bray-Curtis Dissimilarity

 
 
 

Interactive 3D PCoA Plots - Euclidean Distance

 
 
 

Interactive 3D PCoA Plots - Correlation Coefficients

 
 
 

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.all: Cavity vs NoCavity
 
 

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.all: Cavity vs NoCavity
 
 
 

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.

 
all: Cavity vs NoCavity
 
 
 
 
 
 
 

XI. Analysis - Heatmap Profile

 

Species vs Sample Abundance Heatmap for All Samples

 
 
 

Heatmaps for Individual Comparisons

 
Comparison No.Comparison NameFamily LevelGenus LevelSpecies Level
Comparison 1all: Cavity vs NoCavityPDFSVGPDFSVGPDFSVG
 
 

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

 
 

 
 

XIII. Disclaimer

The results of this analysis are for research purpose only. They are not intended to diagnose, treat, cure, or prevent any disease. Forsyth and FOMC are not responsible for use of information provided in this report outside the research area.

 
Copyright FOMC 2021