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
VII.Analysis - Read Taxonomy Assignment
 Taxonomy Barplots
VIII.Analysis - Alpha Diversity
IX.Analysis - Beta Diversity
X.Analysis - Differential Abundance
 ANCOM Result
 LEfSe Result
XI.Analysis - Heatmap Profile
XII.Analysis - Network Association
(Click to navigate)

FOMC Service Report

16S rRNA Gene V1V3 Amplicon Sequencing

Version V1.1

The Forsyth Institute, Cambridge, MA, USA
March 30, 2021

Project ID: FOMC3969

I. Project Summary

Project FOMC3969 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.

Bioinformatics analysis service was not requested, however we still provide the sequence data quality trimming, noise-filtering, pair merging, as well as chimera filtering for the sequences, using the DADA2 denoising algorithm and pipeline. The denoised, merged and chimera-free ASV (amplicon sequence variants) sequences allow you to perform downstream analyses such as taxonomy assignment, diversity analysis and differential abundance analysis. If you need us help with these downstream bioinformatics analysis please contact us.

 

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 (service not requested)
☐10.Bioinformatics Analysis - Alpha Diversity Analysis (service not requested)
☐11.Bioinformatics Analysis - Beta Diversity Analysis (service not requested)
☐12.Bioinformatics Analysis - Differential Abundance Analysis (service not requested)
☐13.Bioinformatics Analysis - Heatmap Profile (service not requested)
☐14.Bioinformatics Analysis - Network Association (service not requested)
 

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.

Absolute Abundance Quantification*: A quantitative real-time PCR was set up with a standard curve. The standard curve was made with plasmid DNA containing one copy of the 16S gene and one copy of the fungal ITS2 region prepared in 10-fold serial dilutions. The primers used were the same as those used in Targeted Library Preparation. The equation generated by the plasmid DNA standard curve was used to calculate the number of gene copies in the reaction for each sample. The PCR input volume (2 µl) was used to calculate the number of gene copies per microliter in each DNA sample.
The number of genome copies per microliter DNA sample was calculated by dividing the gene copy number by an assumed number of gene copies per genome. The value used for 16S copies per genome is 4. The value used for ITS copies per genome is 200. The amount of DNA per microliter DNA sample was calculated using an assumed genome size of 4.64 x 106 bp, the genome size of Escherichia coli, for 16S samples, or an assumed genome size of 1.20 x 107 bp, the genome size of Saccharomyces cerevisiae, for ITS samples. This calculation is shown below:

Calculated Total DNA = Calculated Total Genome Copies × Assumed Genome Size (4.64 × 106 bp) ×
Average Molecular Weight of a DNA bp (660 g/mole/bp) ÷ Avogadro’s Number (6.022 x 1023/mole)


* Absolute Abundance Quantification is only available for 16S and ITS analyses.

The absolute abundance standard curve data can be viewed in Excel here:

The absolute abundance standard curve is shown below:

Absolute Abundance Standard Curve

 

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 IDRead 1 File NameR1 Read Count


S100zr3969_100V3V4_R1.fastq.gz57686


S101zr3969_101V3V4_R1.fastq.gz64213


S102zr3969_102V3V4_R1.fastq.gz57300


S103zr3969_103V3V4_R1.fastq.gz51363


S104zr3969_104V3V4_R1.fastq.gz51194


S105zr3969_105V3V4_R1.fastq.gz45951


S106zr3969_106V3V4_R1.fastq.gz58614


S107zr3969_107V3V4_R1.fastq.gz51632


S108zr3969_108V3V4_R1.fastq.gz44778


S109zr3969_109V3V4_R1.fastq.gz49961


S010zr3969_10V3V4_R1.fastq.gz35651


S110zr3969_110V3V4_R1.fastq.gz58633


S111zr3969_111V3V4_R1.fastq.gz44310


S112zr3969_112V3V4_R1.fastq.gz49783


S113zr3969_113V3V4_R1.fastq.gz46838


S114zr3969_114V3V4_R1.fastq.gz47509


S115zr3969_115V3V4_R1.fastq.gz46061


S116zr3969_116V3V4_R1.fastq.gz53797


S117zr3969_117V3V4_R1.fastq.gz45489


S118zr3969_118V3V4_R1.fastq.gz56119


S119zr3969_119V3V4_R1.fastq.gz45726


S011zr3969_11V3V4_R1.fastq.gz31501


S120zr3969_120V3V4_R1.fastq.gz35338


S121zr3969_121V3V4_R1.fastq.gz41004


S122zr3969_122V3V4_R1.fastq.gz54573


S123zr3969_123V3V4_R1.fastq.gz44927


S124zr3969_124V3V4_R1.fastq.gz54265


S125zr3969_125V3V4_R1.fastq.gz40743


S126zr3969_126V3V4_R1.fastq.gz52825


S127zr3969_127V3V4_R1.fastq.gz50548


S128zr3969_128V3V4_R1.fastq.gz58537


S129zr3969_129V3V4_R1.fastq.gz52716


S012zr3969_12V3V4_R1.fastq.gz37072


S130zr3969_130V3V4_R1.fastq.gz56772


S131zr3969_131V3V4_R1.fastq.gz50817


S132zr3969_132V3V4_R1.fastq.gz56193


S133zr3969_133V3V4_R1.fastq.gz66032


S134zr3969_134V3V4_R1.fastq.gz66323


S135zr3969_135V3V4_R1.fastq.gz54813


S136zr3969_136V3V4_R1.fastq.gz58607


S137zr3969_137V3V4_R1.fastq.gz48068


S138zr3969_138V3V4_R1.fastq.gz51823


S139zr3969_139V3V4_R1.fastq.gz53430


S013zr3969_13V3V4_R1.fastq.gz34157


S140zr3969_140V3V4_R1.fastq.gz55720


S141zr3969_141V3V4_R1.fastq.gz47995


S142zr3969_142V3V4_R1.fastq.gz52547


S143zr3969_143V3V4_R1.fastq.gz51597


S144zr3969_144V3V4_R1.fastq.gz55689


S145zr3969_145V3V4_R1.fastq.gz50968


S146zr3969_146V3V4_R1.fastq.gz44903


S147zr3969_147V3V4_R1.fastq.gz37927


S148zr3969_148V3V4_R1.fastq.gz49600


S149zr3969_149V3V4_R1.fastq.gz47608


S014zr3969_14V3V4_R1.fastq.gz40213


S150zr3969_150V3V4_R1.fastq.gz48138


S151zr3969_151V3V4_R1.fastq.gz46628


S152zr3969_152V3V4_R1.fastq.gz52770


S153zr3969_153V3V4_R1.fastq.gz58216


S154zr3969_154V3V4_R1.fastq.gz53012


S155zr3969_155V3V4_R1.fastq.gz52276


S156zr3969_156V3V4_R1.fastq.gz51293


S157zr3969_157V3V4_R1.fastq.gz38080


S158zr3969_158V3V4_R1.fastq.gz60956


S159zr3969_159V3V4_R1.fastq.gz47558


S015zr3969_15V3V4_R1.fastq.gz31833


S160zr3969_160V3V4_R1.fastq.gz50363


S161zr3969_161V3V4_R1.fastq.gz49670


S162zr3969_162V3V4_R1.fastq.gz56932


S163zr3969_163V3V4_R1.fastq.gz50274


S164zr3969_164V3V4_R1.fastq.gz53183


S165zr3969_165V3V4_R1.fastq.gz65877


S166zr3969_166V3V4_R1.fastq.gz46466


S167zr3969_167V3V4_R1.fastq.gz39934


S168zr3969_168V3V4_R1.fastq.gz54999


S169zr3969_169V3V4_R1.fastq.gz47943


S016zr3969_16V3V4_R1.fastq.gz40929


S170zr3969_170V3V4_R1.fastq.gz44212


S171zr3969_171V3V4_R1.fastq.gz36601


S172zr3969_172V3V4_R1.fastq.gz40639


S173zr3969_173V3V4_R1.fastq.gz57180


S174zr3969_174V3V4_R1.fastq.gz52818


S175zr3969_175V3V4_R1.fastq.gz55930


S176zr3969_176V3V4_R1.fastq.gz57383


S177zr3969_177V3V4_R1.fastq.gz54514


S178zr3969_178V3V4_R1.fastq.gz63196


S179zr3969_179V3V4_R1.fastq.gz32851


S017zr3969_17V3V4_R1.fastq.gz26838


S180zr3969_180V3V4_R1.fastq.gz41633


S181zr3969_181V3V4_R1.fastq.gz66099


S182zr3969_182V3V4_R1.fastq.gz59228


S183zr3969_183V3V4_R1.fastq.gz39017


S184zr3969_184V3V4_R1.fastq.gz42880


S185zr3969_185V3V4_R1.fastq.gz41603


S186zr3969_186V3V4_R1.fastq.gz34376


S187zr3969_187V3V4_R1.fastq.gz41611


S188zr3969_188V3V4_R1.fastq.gz51972


S189zr3969_189V3V4_R1.fastq.gz46941


S018zr3969_18V3V4_R1.fastq.gz32085


S190zr3969_190V3V4_R1.fastq.gz38301


S191zr3969_191V3V4_R1.fastq.gz67451


S192zr3969_192V3V4_R1.fastq.gz68273


S193zr3969_193V3V4_R1.fastq.gz64174


S194zr3969_194V3V4_R1.fastq.gz58156


S195zr3969_195V3V4_R1.fastq.gz56109


S196zr3969_196V3V4_R1.fastq.gz42529


S197zr3969_197V3V4_R1.fastq.gz45713


S198zr3969_198V3V4_R1.fastq.gz52031


S199zr3969_199V3V4_R1.fastq.gz59612


S019zr3969_19V3V4_R1.fastq.gz24967


S001zr3969_1V3V4_R1.fastq.gz29337


S200zr3969_200V3V4_R1.fastq.gz51467


S201zr3969_201V3V4_R1.fastq.gz53096


S202zr3969_202V3V4_R1.fastq.gz57595


S203zr3969_203V3V4_R1.fastq.gz51017


S204zr3969_204V3V4_R1.fastq.gz62912


S205zr3969_205V3V4_R1.fastq.gz46372


S206zr3969_206V3V4_R1.fastq.gz47607


S207zr3969_207V3V4_R1.fastq.gz58703


S208zr3969_208V3V4_R1.fastq.gz46178


S209zr3969_209V3V4_R1.fastq.gz49425


S020zr3969_20V3V4_R1.fastq.gz26467


S210zr3969_210V3V4_R1.fastq.gz47924


S211zr3969_211V3V4_R1.fastq.gz58269


S212zr3969_212V3V4_R1.fastq.gz52294


S213zr3969_213V3V4_R1.fastq.gz47759


S214zr3969_214V3V4_R1.fastq.gz51271


S215zr3969_215V3V4_R1.fastq.gz54279


S216zr3969_216V3V4_R1.fastq.gz43200


S217zr3969_217V3V4_R1.fastq.gz42887


S218zr3969_218V3V4_R1.fastq.gz54018


S219zr3969_219V3V4_R1.fastq.gz48233


S021zr3969_21V3V4_R1.fastq.gz19069


S220zr3969_220V3V4_R1.fastq.gz46877


S221zr3969_221V3V4_R1.fastq.gz48579


S222zr3969_222V3V4_R1.fastq.gz60803


S223zr3969_223V3V4_R1.fastq.gz75296


S224zr3969_224V3V4_R1.fastq.gz47004


S225zr3969_225V3V4_R1.fastq.gz63569


S226zr3969_226V3V4_R1.fastq.gz54171


S227zr3969_227V3V4_R1.fastq.gz55287


S228zr3969_228V3V4_R1.fastq.gz77356


S229zr3969_229V3V4_R1.fastq.gz49436


S022zr3969_22V3V4_R1.fastq.gz38424


S230zr3969_230V3V4_R1.fastq.gz56393


S231zr3969_231V3V4_R1.fastq.gz57384


S232zr3969_232V3V4_R1.fastq.gz49383


S233zr3969_233V3V4_R1.fastq.gz71153


S234zr3969_234V3V4_R1.fastq.gz49111


S235zr3969_235V3V4_R1.fastq.gz54257


S236zr3969_236V3V4_R1.fastq.gz68153


S237zr3969_237V3V4_R1.fastq.gz51559


S238zr3969_238V3V4_R1.fastq.gz50031


S239zr3969_239V3V4_R1.fastq.gz36482


S023zr3969_23V3V4_R1.fastq.gz28731


S240zr3969_240V3V4_R1.fastq.gz49291


S241zr3969_241V3V4_R1.fastq.gz44366


S242zr3969_242V3V4_R1.fastq.gz49309


S243zr3969_243V3V4_R1.fastq.gz46308


S244zr3969_244V3V4_R1.fastq.gz42626


S245zr3969_245V3V4_R1.fastq.gz39535


S246zr3969_246V3V4_R1.fastq.gz46239


S247zr3969_247V3V4_R1.fastq.gz58091


S248zr3969_248V3V4_R1.fastq.gz49309


S249zr3969_249V3V4_R1.fastq.gz43365


S024zr3969_24V3V4_R1.fastq.gz34616


S250zr3969_250V3V4_R1.fastq.gz47971


S251zr3969_251V3V4_R1.fastq.gz52868


S252zr3969_252V3V4_R1.fastq.gz57570


S253zr3969_253V3V4_R1.fastq.gz50748


S254zr3969_254V3V4_R1.fastq.gz48168


S255zr3969_255V3V4_R1.fastq.gz61936


S256zr3969_256V3V4_R1.fastq.gz50791


S257zr3969_257V3V4_R1.fastq.gz54746


S258zr3969_258V3V4_R1.fastq.gz58128


S259zr3969_259V3V4_R1.fastq.gz55424


S025zr3969_25V3V4_R1.fastq.gz25009


S260zr3969_260V3V4_R1.fastq.gz60352


S261zr3969_261V3V4_R1.fastq.gz42924


S262zr3969_262V3V4_R1.fastq.gz50320


S263zr3969_263V3V4_R1.fastq.gz51439


S264zr3969_264V3V4_R1.fastq.gz40764


S265zr3969_265V3V4_R1.fastq.gz49534


S266zr3969_266V3V4_R1.fastq.gz40610


S267zr3969_267V3V4_R1.fastq.gz50476


S268zr3969_268V3V4_R1.fastq.gz65918


S269zr3969_269V3V4_R1.fastq.gz51437


S026zr3969_26V3V4_R1.fastq.gz28792


S270zr3969_270V3V4_R1.fastq.gz58627


S271zr3969_271V3V4_R1.fastq.gz60329


S272zr3969_272V3V4_R1.fastq.gz54631


S273zr3969_273V3V4_R1.fastq.gz65741


S274zr3969_274V3V4_R1.fastq.gz51977


S275zr3969_275V3V4_R1.fastq.gz54570


S276zr3969_276V3V4_R1.fastq.gz52315


S277zr3969_277V3V4_R1.fastq.gz45171


S278zr3969_278V3V4_R1.fastq.gz42346


S279zr3969_279V3V4_R1.fastq.gz22935


S027zr3969_27V3V4_R1.fastq.gz23076


S280zr3969_280V3V4_R1.fastq.gz40630


S281zr3969_281V3V4_R1.fastq.gz46606


S282zr3969_282V3V4_R1.fastq.gz37014


S283zr3969_283V3V4_R1.fastq.gz33767


S284zr3969_284V3V4_R1.fastq.gz43672


S285zr3969_285V3V4_R1.fastq.gz40662


S286zr3969_286V3V4_R1.fastq.gz44233


S287zr3969_287V3V4_R1.fastq.gz44782


S288zr3969_288V3V4_R1.fastq.gz38373


S289zr3969_289V3V4_R1.fastq.gz48221


S028zr3969_28V3V4_R1.fastq.gz30570


S290zr3969_290V3V4_R1.fastq.gz49571


S291zr3969_291V3V4_R1.fastq.gz54574


S292zr3969_292V3V4_R1.fastq.gz54601


S293zr3969_293V3V4_R1.fastq.gz49871


S294zr3969_294V3V4_R1.fastq.gz43083


S295zr3969_295V3V4_R1.fastq.gz42279


S296zr3969_296V3V4_R1.fastq.gz56385


S297zr3969_297V3V4_R1.fastq.gz45414


S298zr3969_298V3V4_R1.fastq.gz50795


S299zr3969_299V3V4_R1.fastq.gz38622


S029zr3969_29V3V4_R1.fastq.gz28089


S002zr3969_2V3V4_R1.fastq.gz34578


S300zr3969_300V3V4_R1.fastq.gz50782


S301zr3969_301V3V4_R1.fastq.gz59395


S302zr3969_302V3V4_R1.fastq.gz40517


S303zr3969_303V3V4_R1.fastq.gz44647


S304zr3969_304V3V4_R1.fastq.gz40817


S305zr3969_305V3V4_R1.fastq.gz47800


S306zr3969_306V3V4_R1.fastq.gz60196


S307zr3969_307V3V4_R1.fastq.gz39794


S308zr3969_308V3V4_R1.fastq.gz41100


S309zr3969_309V3V4_R1.fastq.gz45986


S030zr3969_30V3V4_R1.fastq.gz37150


S310zr3969_310V3V4_R1.fastq.gz44669


S311zr3969_311V3V4_R1.fastq.gz41889


S312zr3969_312V3V4_R1.fastq.gz48123


S313zr3969_313V3V4_R1.fastq.gz46459


S314zr3969_314V3V4_R1.fastq.gz44808


S315zr3969_315V3V4_R1.fastq.gz37835


S316zr3969_316V3V4_R1.fastq.gz52769


S317zr3969_317V3V4_R1.fastq.gz43577


S318zr3969_318V3V4_R1.fastq.gz44579


S319zr3969_319V3V4_R1.fastq.gz43467


S031zr3969_31V3V4_R1.fastq.gz32013


S320zr3969_320V3V4_R1.fastq.gz49868


S321zr3969_321V3V4_R1.fastq.gz52030


S322zr3969_322V3V4_R1.fastq.gz49082


S323zr3969_323V3V4_R1.fastq.gz55031


S324zr3969_324V3V4_R1.fastq.gz63518


S325zr3969_325V3V4_R1.fastq.gz53196


S326zr3969_326V3V4_R1.fastq.gz54850


S327zr3969_327V3V4_R1.fastq.gz57885


S328zr3969_328V3V4_R1.fastq.gz57875


S329zr3969_329V3V4_R1.fastq.gz60830


S032zr3969_32V3V4_R1.fastq.gz36067


S330zr3969_330V3V4_R1.fastq.gz63088


S331zr3969_331V3V4_R1.fastq.gz32616


S332zr3969_332V3V4_R1.fastq.gz43994


S333zr3969_333V3V4_R1.fastq.gz38912


S334zr3969_334V3V4_R1.fastq.gz45933


S335zr3969_335V3V4_R1.fastq.gz40273


S336zr3969_336V3V4_R1.fastq.gz41550


S337zr3969_337V3V4_R1.fastq.gz47445


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S009zr3969_9V3V4_R1.fastq.gz29954

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 merging 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 analysis 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/R2281271261251241231
32142.34%54.39%54.26%54.24%54.29%54.18%
31143.40%58.15%58.00%57.96%57.87%57.81%
30143.60%57.98%58.00%58.20%58.11%58.12%
29144.37%58.53%58.60%58.74%58.92%58.74%
28144.38%58.58%58.74%59.02%59.03%58.91%
27144.49%58.78%58.89%59.13%59.26%59.06%

Based on the above result, the trim length combination of R1 = 271 bases and R2 = 241 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 IDF3969.S001F3969.S002F3969.S003F3969.S004F3969.S005F3969.S006F3969.S007F3969.S008F3969.S009F3969.S010F3969.S011F3969.S012F3969.S013F3969.S014F3969.S015F3969.S016F3969.S017F3969.S018F3969.S019F3969.S020F3969.S021F3969.S022F3969.S023F3969.S024F3969.S025F3969.S026F3969.S027F3969.S028F3969.S029F3969.S030F3969.S031F3969.S032F3969.S033F3969.S034F3969.S035F3969.S036F3969.S037F3969.S038F3969.S039F3969.S040F3969.S041F3969.S042F3969.S043F3969.S044F3969.S045F3969.S046F3969.S047F3969.S048F3969.S049F3969.S050F3969.S051F3969.S052F3969.S053F3969.S054F3969.S055F3969.S056F3969.S057F3969.S058F3969.S059F3969.S060F3969.S061F3969.S062F3969.S063F3969.S064F3969.S065F3969.S066F3969.S067F3969.S068F3969.S069F3969.S070F3969.S071F3969.S072F3969.S073F3969.S074F3969.S075F3969.S076F3969.S077F3969.S078F3969.S079F3969.S080F3969.S081F3969.S082F3969.S083F3969.S084F3969.S085F3969.S086F3969.S087F3969.S088F3969.S089F3969.S090F3969.S091F3969.S092F3969.S093F3969.S094F3969.S095F3969.S096F3969.S097F3969.S098F3969.S099F3969.S100F3969.S101F3969.S102F3969.S103F3969.S104F3969.S105F3969.S106F3969.S107F3969.S108F3969.S109F3969.S110F3969.S111F3969.S112F3969.S113F3969.S114F3969.S115F3969.S116F3969.S117F3969.S118F3969.S119F3969.S120F3969.S121F3969.S122F3969.S123F3969.S124F3969.S125F3969.S126F3969.S127F3969.S128F3969.S129F3969.S130F3969.S131F3969.S132F3969.S133F3969.S134F3969.S135F3969.S136F3969.S137F3969.S138F3969.S139F3969.S140F3969.S141F3969.S142F3969.S143F3969.S144F3969.S145F3969.S146F3969.S147F3969.S148F3969.S149F3969.S150F3969.S151F3969.S152F3969.S153F3969.S154F3969.S155F3969.S156F3969.S157F3969.S158F3969.S159F3969.S160F3969.S161F3969.S162F3969.S163F3969.S164F3969.S165F3969.S166F3969.S167F3969.S168F3969.S169F3969.S170F3969.S171F3969.S172F3969.S173F3969.S174F3969.S175F3969.S176F3969.S177F3969.S178F3969.S179F3969.S180F3969.S181F3969.S182F3969.S183F3969.S184F3969.S185F3969.S186F3969.S187F3969.S188F3969.S189F3969.S190F3969.S191F3969.S192F3969.S193F3969.S194F3969.S195F3969.S196F3969.S197F3969.S198F3969.S199F3969.S200F3969.S201F3969.S202F3969.S203F3969.S204F3969.S205F3969.S206F3969.S207F3969.S208F3969.S209F3969.S210F3969.S211F3969.S212F3969.S213F3969.S214F3969.S215F3969.S216F3969.S217F3969.S218F3969.S219F3969.S220F3969.S221F3969.S222F3969.S223F3969.S224F3969.S225F3969.S226F3969.S227F3969.S228F3969.S229F3969.S230F3969.S231F3969.S232F3969.S233F3969.S234F3969.S235F3969.S236F3969.S237F3969.S238F3969.S239F3969.S240F3969.S241F3969.S242F3969.S243F3969.S244F3969.S245F3969.S246F3969.S247F3969.S248F3969.S249F3969.S250F3969.S251F3969.S252F3969.S253F3969.S254F3969.S255F3969.S256F3969.S257F3969.S258F3969.S259F3969.S260F3969.S261F3969.S262F3969.S263F3969.S264F3969.S265F3969.S266F3969.S267F3969.S268F3969.S269F3969.S270F3969.S271F3969.S272F3969.S273F3969.S274F3969.S275F3969.S276F3969.S277F3969.S278F3969.S279F3969.S280F3969.S281F3969.S282F3969.S283F3969.S284F3969.S285F3969.S286F3969.S287F3969.S288F3969.S289F3969.S290F3969.S291F3969.S292F3969.S293F3969.S294F3969.S295F3969.S296F3969.S297F3969.S298F3969.S299F3969.S300F3969.S301F3969.S302F3969.S303F3969.S304F3969.S305F3969.S306F3969.S307F3969.S308F3969.S309F3969.S310F3969.S311F3969.S312F3969.S313F3969.S314F3969.S315F3969.S316F3969.S317F3969.S318F3969.S319F3969.S320F3969.S321F3969.S322F3969.S323F3969.S324F3969.S325F3969.S326F3969.S327F3969.S328F3969.S329F3969.S330F3969.S331F3969.S332F3969.S333F3969.S334F3969.S335F3969.S336F3969.S337F3969.S338F3969.S339F3969.S340F3969.S341F3969.S342F3969.S343Row SumPercentage
input29,33734,57828,94632,51427,13034,73728,58237,16829,95435,65131,50137,07234,15740,21331,83340,92926,83832,08524,96726,46719,06938,42428,73134,61625,00928,79223,07630,57028,08937,15032,01336,06726,39237,49331,09330,99133,13734,72635,24834,91836,17935,69233,95437,43333,82638,92631,50037,05223,48222,93021,27721,41021,04026,46423,66928,25130,50533,94531,99530,46833,13134,88339,68435,68728,13734,28127,74861,72029,65134,59931,28533,79629,61531,15722,28226,84823,77031,93424,66530,25629,27232,74629,91132,48126,72237,85926,05937,17329,91938,49528,19538,78228,48138,32159,23962,65166,42148,21046,98457,68664,21357,30051,36351,19445,95158,61451,63244,77849,96158,63344,31049,78346,83847,50946,06153,79745,48956,11945,72635,33841,00454,57344,92754,26540,74352,82550,54858,53752,71656,77250,81756,19366,03266,32354,81358,60748,06851,82353,43055,72047,99552,54751,59755,68950,96844,90337,92749,60047,60848,13846,62852,77058,21653,01252,27651,29338,08060,95647,55850,36349,67056,93250,27453,18365,87746,46639,93454,99947,94344,21236,60140,63957,18052,81855,93057,38354,51463,19632,85141,63366,09959,22839,01742,88041,60334,37641,61151,97246,94138,30167,45168,27364,17458,15656,10942,52945,71352,03159,61251,46753,09657,59551,01762,91246,37247,60758,70346,17849,42547,92458,26952,29447,75951,27154,27943,20042,88754,01848,23346,87748,57960,80375,29647,00463,56954,17155,28777,35649,43656,39357,38449,38371,15349,11154,25768,15351,55950,03136,48249,29144,36649,30946,30842,62639,53546,23958,09149,30943,36547,97152,86857,57050,74848,16861,93650,79154,74658,12855,42460,35242,92450,32051,43940,76449,53440,61050,47665,91851,43758,62760,32954,63165,74151,97754,57052,31545,17142,34622,93540,63046,60637,01433,76743,67240,66244,23344,78238,37348,22149,57154,57454,60149,87143,08342,27956,38545,41450,79538,62250,78259,39540,51744,64740,81747,80060,19639,79441,10045,98644,66941,88948,12346,45944,80837,83552,76943,57744,57943,46749,86852,03049,08255,03163,51853,19654,85057,88557,87560,83063,08832,61643,99438,91245,93340,27341,55047,44551,30149,30347,23058,04472,33856,61315,591,941100.00%
filtered26,40031,44026,21729,88423,53331,10524,93233,02627,56632,38228,55333,18031,58136,30528,68236,46524,57829,11022,09823,62316,45134,87324,98730,72422,69026,27720,69127,60325,46133,77828,74832,20423,38334,07028,25927,84930,27031,23331,70331,63732,38832,64130,78434,29730,46035,09527,19033,28321,29820,74019,07619,35118,94724,00421,48225,39426,98130,52828,66327,08029,63329,91736,06631,70224,97830,80224,52155,60226,91930,62227,94830,06926,83328,53719,69123,72521,27028,73421,77726,91426,23929,79527,05029,64324,02334,47923,08433,53726,69934,54725,57735,19425,54234,67452,18056,30859,02341,50842,55751,00858,32550,76145,25945,09139,98052,35346,25439,80044,80953,01139,00043,51341,04041,88040,68948,35739,80650,18740,56430,14235,05147,97939,80747,93835,36047,29244,28350,97346,10349,57844,41449,57158,98159,47748,15552,75941,44343,18947,63549,13341,20946,08045,95449,85644,83438,34632,41443,97542,37741,81940,54647,00252,85148,02346,38544,60532,53855,37242,29945,85942,97851,25346,00347,86958,66240,53534,82649,61042,72038,37233,35935,36451,10847,02149,92750,79947,74356,46127,39835,69759,08652,12834,52538,56337,17430,37737,44346,62941,67334,15559,64661,05557,53552,42849,25536,19440,53145,59752,35545,87747,87251,23645,55855,93940,52741,42751,46140,98544,23042,82552,02646,25642,55946,19347,35338,23737,57348,66342,21741,20542,17554,42567,75541,04256,10949,14848,28769,39544,86350,25251,00543,17665,16644,97849,05460,90946,03144,78432,24343,34140,41243,16339,85538,55835,22641,59251,05444,86439,08942,09247,10051,96344,89542,75454,57344,76048,60052,78450,05054,04938,04744,90945,88135,29044,14336,60244,76759,05446,58152,31952,41047,62159,44447,51947,33146,26540,43438,31619,26636,24041,46633,65429,58838,87135,72638,85540,35834,42242,45343,74148,39149,42444,00138,55836,71250,91840,88544,68134,12945,40152,95836,35439,08336,56641,62053,58035,72635,73240,47340,17936,97441,81341,40439,10934,02747,23639,02939,28638,81144,97145,27543,14249,04656,94148,24348,85351,53551,57554,71555,89428,15339,34934,26241,48636,04836,85542,57145,77242,72841,71051,34864,80950,21713,870,42188.96%
denoisedF25,60129,92425,83329,68822,73929,61323,99432,12427,23831,82827,20532,04231,07935,20027,18935,05723,44427,75721,19521,95516,19733,46924,18429,15721,39225,03819,83226,33723,80132,77427,95330,48422,58433,44127,45726,32029,12229,65230,40330,06430,99931,56430,30733,62629,14833,40026,75932,56020,56919,90618,18718,48218,06423,32720,64724,35725,91929,98126,92126,06628,92628,23035,53830,47023,12529,53823,36253,64426,62829,05126,17829,30326,34327,46119,20923,11419,91527,60420,37526,01624,72229,30126,41027,98023,76334,27622,57932,88925,22733,26324,49733,30124,23033,52749,81155,26256,20540,26841,85848,51157,65250,21542,61943,02138,07750,75443,51338,39843,08452,28737,76040,94139,01639,67038,83447,07437,87349,07639,50029,49134,37945,21338,07845,61033,22045,19542,13149,60344,25746,97741,86746,67958,08757,55446,48550,74340,09140,64145,63946,87839,31844,09144,99447,75342,58736,72530,87642,48240,16239,53239,16745,87652,16845,93944,89943,49731,19854,55140,65644,89142,00750,36645,35346,34258,23639,77933,44049,07640,64236,59932,94033,88750,04245,89448,16949,65045,73255,07526,03834,34958,58349,70133,43036,81535,47329,62934,90445,15739,57833,06057,97258,74255,76650,59947,17635,40538,57144,39550,66944,10546,20548,83544,24652,66438,64039,89949,19039,99242,15540,58150,19244,41242,03644,81545,74837,39436,40446,92840,36839,80939,96452,75766,72639,53453,84147,56147,21768,62143,44447,99849,88341,95164,80843,67747,56958,74244,49743,65131,39441,02840,32541,64538,69137,43834,02140,68449,14844,29838,05341,07945,29250,05042,84041,36552,03643,38048,16052,38448,77352,30737,23242,66445,01134,08742,71835,33844,08956,41145,16150,35251,20045,51357,94346,26045,37544,95538,16137,26018,51834,97340,04532,55728,55037,30134,59138,10139,25233,67041,28842,18847,06748,00842,74537,42535,41850,40739,85643,52033,02344,12851,82135,60938,06535,82040,08152,17934,35234,25839,08339,16335,62140,18140,90538,66432,99545,87337,73438,29937,52443,51243,85541,34247,76255,81147,05246,99450,06550,12553,81354,79527,57337,90732,91940,58535,74234,90840,91545,26441,17439,40049,60563,08849,53013,407,62485.99%
denoisedR25,71030,20325,97929,38322,68330,09124,02532,20326,80331,69427,92432,07731,21235,06227,78035,26023,69727,64221,37822,66816,34133,79424,12129,69322,07925,46419,95326,42424,42232,68527,80731,28622,68133,13427,48527,06729,60929,85530,23930,30731,32531,31029,73133,36729,19133,77326,71132,07120,36919,91418,28918,59718,10223,38020,37624,43926,02629,57127,36226,17029,11928,73935,41930,23324,10829,60823,66954,18926,43329,46527,02029,11925,95927,38618,93123,30020,50427,43121,08126,20125,44429,29526,18828,27423,76734,06222,71333,09325,71333,39124,65633,70424,30133,35850,29454,54257,29540,38841,95749,10457,86650,05743,60843,69538,39650,82344,31538,67743,90950,70637,87442,31839,21140,48439,26447,14738,15749,48539,74129,15834,44346,01438,17146,18734,15145,55542,97449,76944,69547,78143,01348,08757,79657,36146,69751,09440,25940,49146,27347,22639,57044,29245,22948,11943,39037,09931,15342,71340,48040,31139,27246,21051,86146,55744,53143,62531,57454,54640,70844,66841,78850,12645,26346,20157,98839,74233,44448,94741,43637,01033,19734,14950,33745,91948,53849,71545,83654,77026,58134,42158,00249,96833,25736,99835,68729,39436,02845,47840,12732,84558,56858,66956,21950,76647,29535,06039,44143,97450,70444,69246,15349,62044,29053,83339,28940,13549,66240,12542,53441,16049,99144,53341,35345,09946,15536,92436,39047,19440,40439,69940,59753,26366,84339,85753,97148,24547,26268,24944,20948,74649,45042,16264,43944,23247,98758,91444,94643,39831,73941,58640,32741,98038,45737,81834,26440,92149,26844,59338,36440,73745,81950,03143,80441,15052,86543,81548,25551,91148,92152,24236,62643,00244,58434,49742,46935,70744,24356,90945,24651,30450,66746,34558,39146,85146,13145,13739,11736,73618,74135,36340,07232,87528,86237,72234,26137,95539,38133,75141,34942,33347,10348,56542,62637,39535,68750,33940,28043,31533,38244,34152,10935,69337,86235,70740,24351,68234,85434,72239,00039,54536,00140,29040,76138,85233,32146,14238,23238,16837,48343,59543,95541,29147,59455,60947,11947,24749,89849,86753,53254,42927,35938,29333,03940,66635,32035,52540,91844,97441,23140,47549,37763,84048,97413,469,49786.39%
merged24,23228,02925,41328,88220,57828,12122,12831,12125,70230,26425,80430,54530,38133,87525,51133,49021,40725,93119,22019,07415,64231,83422,53526,34719,00823,43018,12523,86622,02531,54726,47828,46820,86732,13626,47224,31627,90427,42328,85227,54427,88828,77129,21032,47426,55831,28025,92330,02019,05718,52116,70016,91516,76922,24718,63322,40624,16027,78925,14723,27828,18525,12833,30427,92620,49227,55421,12551,08626,08826,51524,67627,64225,12525,41818,03721,84418,04625,67418,30524,53922,35128,16424,97325,30123,48033,74821,85232,17922,64931,48723,12330,42621,89530,53045,26052,43053,72437,35340,92143,82556,68349,32638,63840,57934,38646,59840,48035,99540,64347,91335,41437,39335,81636,69835,68345,12733,99147,12737,63427,86533,18742,65535,89843,26229,29342,80838,43347,05442,09542,08538,80642,87856,60554,15543,62447,25037,36438,38043,00741,86335,75240,31244,10545,16538,89034,58027,82940,92337,61635,32236,80343,47650,18643,55741,46040,66129,69353,28136,72042,74839,59448,29442,71843,92557,43838,22130,37548,14937,67533,21132,78831,62848,93344,07443,71946,94341,41852,38224,18931,87257,49944,93730,26534,04832,33728,08932,51242,80837,29930,89455,58054,35453,54047,00943,52733,96536,62542,03048,03141,46543,33545,40841,77748,77135,14937,38045,02438,41739,28936,75347,25640,37440,36742,31343,10335,13434,14744,65837,58237,25336,52550,05564,39937,59449,56744,74945,26666,54340,40544,38746,73240,01563,38040,83543,53054,29042,11441,56129,70237,08740,30939,41936,33734,78431,26238,85844,19843,26835,75238,00441,61346,56841,19939,10848,03541,64647,14351,05245,51847,29334,15539,30843,08832,75238,90433,03243,29551,56641,97447,51047,59942,54556,41643,39141,60242,07135,46834,80816,80832,69737,92129,81926,81534,60232,53036,52836,19131,68638,87839,44645,05245,03440,52634,93933,15249,60737,46740,14230,75942,57150,51833,22435,61533,36635,79549,71432,23732,06636,21236,82933,08236,88439,64138,01331,00242,76635,18935,86035,01740,66641,19637,66944,58153,56044,06643,71347,53646,35051,53351,76126,19435,76329,77239,20334,39031,56336,88043,86137,09436,68046,23361,43047,59512,587,34880.73%
nonchim6,0037,1793,1513,0804,7237,0935,5375,8053,0856,6018,5196,0334,5016,3497,2696,6326,3945,8865,5569,1443,4987,2124,48410,2887,0008,7328,0016,06010,0635,9855,58712,1776,7614,7183,9588,9548,4939,8696,6039,3649,1166,4234,2246,6046,9077,8233,4124,8283,5775,5935,4344,5875,0824,4844,9738,2115,7903,4206,3126,6145,4038,9873,9954,9818,72710,1838,3758,6862,58510,22210,7446,2173,6375,0913,4425,4007,9935,2618,0776,3458,0505,0566,0028,7624,0633,2864,5825,9189,5006,3925,9097,3775,4247,66314,8706,23710,9737,4375,85214,6346,3902,90215,71913,84710,4665,6089,3538,37510,3344,7176,89912,3128,51410,12114,1448,46411,3656,7556,3335,0525,5179,0778,2609,44410,6587,85211,7219,2485,73415,69715,43415,7938,6898,63413,5138,6637,2607,20610,47410,24010,87210,9096,1739,68118,3708,8689,2426,6277,37314,3077,17310,1375,4089,4079,6047,2347,5586,3839,2036,3787,1946,3828,1067,0764,4877,0598,5276,77111,20110,1166,11010,1277,8238,61412,4879,13214,6386,3367,3617,4804,82511,8208,2887,7397,9724,85711,6698,1748,5227,90710,39713,9847,3207,65012,5415,1149,2776,9778,89711,1047,12513,53510,50914,96710,8138,85015,3076,9658,72113,6388,23311,0674,6429,99212,7026,8759,73910,7567,1529,5937,91811,3527,7317,30112,67510,9299,9465,1756,59513,6247,86410,0634,2666,59010,55112,7807,7445,0294,01514,6483,01911,1317,6996,7938,0996,44213,3031,6046,9349,05610,5627,37911,3999,00115,7198,7276,9465,5518,96610,8027,69611,6955,5986,54811,7948,5775,91411,2087,36311,9947,57913,2797,3407,83812,1647,53614,2855,1782,7949,4367,3106,8656,2168,7336,2005,4258,5645,25510,30311,96110,3809,68110,3237,4757,5315,6027,8759,5746,6439,3417,3695,6069,1146,17011,3088,5978,03710,6505,8857,24611,08710,5376,1706,7245,3139,5498,3746,3176,0228,48610,6209,41710,9026,1935,89812,2708,69711,5136,9398,4013,9848,6798,1135,4124,22313,13012,3425,15910,48911,5219,8588,5946,3942,803,34217.98%

This table can be downloaded as an Excel table below:

 

5. DADA2 Amplicon Sequence Variants (ASVs). A total of 59050 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:

 
 
 
 

VII. Analysis - Read Taxonomy Assignment

 
Service not requested
 

VIII. Analysis - Alpha Diversity

 
Service not requested
 

IX. Analysis - RBeta Diversity

 
Service not requested
 

X. Analysis - Differential Abundance

 
Service not requested
 

XI. Analysis - Heatmap Profile

 
Service not requested
 

XII. Analysis - Network Association

 
Service not requested
 
Copyright FOMC 2021