Cited by 587Gut microbiota and healthGenomics and Phylogenetic StudiesMicrobial Community Ecology and Physiology

The DOI matches its Crossref record; 64 of the 64 references it cites were found in a registry or an index; none of the 30 sources its claims cite is checked for support yet.

Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium

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The Microbiome Quality Control Project Consortium, Sinha, Rashmi, Abu-Ali, Galeb, Vogtmann, Emily, Fodor, Anthony A, Ren, Boyu, Amir, Amnon, Schwager, Emma, Crabtree, Jonathan, Ma, Siyuan, Abnet, Christian C, Knight, Rob, White, Owen, Huttenhower, Curtis·2017·Nature Biotechnology

DOI 10.1038/nbt.3981PMC PMC5839636

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Abstract

In order for human microbiome studies to translate into actionable outcomes for health, meta-analysis of reproducible data from population-scale cohorts is needed. Achieving sufficient reproducibility in microbiome research has proven challenging. We report a baseline investigation of variability in taxonomic profiling for the Microbiome Quality Control (MBQC) project baseline study (MBQC-base). Blinded specimen sets from human stool, chemostats, and artificial microbial communities were sequenced by 15 laboratories and analyzed using nine bioinformatics protocols. Variability depended most on biospecimen type and origin, followed by DNA extraction, sample handling environment, and bioinformatics. Analysis of artificial community specimens revealed differences in extraction efficiency and bioinformatic classification. These results may guide researchers in experimental design choices for gut microbiome studies.

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Crossref · 10.1038/nbt.3981

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It may state two separate claims as one. 63
Probably not a substantial claim. 45
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“At least one OTU was reported in 342 of these samples, with the observed number ranging from 1 to ~130 at low (1,000 sequences) rarefaction and almost 200 in all sequences”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Results > Negative controls
Load-bearingStrength:plain· not yet calibratedn = 342
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes
It may state two separate claims as one.
- Rests on an assumption: Negative control buffer aliquots are expected to contain no microbial DNA or OTUs, so any OTUs detected in them indicate contamination.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Beta diversities between locally- versus centrally-extracted samples were specimen-specific, with some samples showing little effect and others differing significantly primarily based on this single handling protocol variable”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Results > Variation due to extraction protocol
Load-bearingStrength:plain· not yet calibratedCausal:direct· not yet calibratedn = 22
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes
It may state two separate claims as one.
- Rests on an assumption: Comparing centrally extracted DNA with locally extracted DNA isolates the effect of DNA extraction from PCR and sequencing effects.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“The most abundant contaminant OTU was an unidentified Bacteroides at 1.6 s.d. 7.0%, which was detected in 22.6% of negative control samples and was also abundant in stool samples (average 1.2 s.d. 3.7%).”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Results > Negative controls
Strength:plain· not yet calibratedn = 345
Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passes
It may state two separate claims as one.
- Rests on an assumption: Negative control buffer aliquots are expected to contain no microbial DNA or OTUs, so any OTUs detected in them indicate contamination.
In the paper
“this experiment supported the expected ground truth and further confirmed the variability of laboratory and bioinformatics protocols when reading out even these low-complexity communities.”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Results > Positive controls
What the sources say
- Not checked yetcites [22] Segata, N. et al. Metagenomic microbial community profiling using unique clade-specific marker genes. Nat. Methods 9, 811–814 (2012). source
Load-bearingStrength:plain· not yet calibratedCausal:correlational· not yet calibratedn = 6
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes
It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Of the top 100 most abundant false positive Greengenes identifiers (average relative abundances ranging from 0.2% to 1.5%), 34% were from genera Bacteroides; Faecalibacterium prausnitzii also comprised 9% of the top 100 IDs, genus Pseudomonas 7%, and family Enterobacteriaceae 6%”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Results > Negative controls
Strength:plain· not yet calibratedn = 345
Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passes
It may state two separate claims as one.
- Rests on an assumption: Negative control buffer aliquots are expected to contain no microbial DNA or OTUs, so any OTUs detected in them indicate contamination.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Sample handling and bioinformatics protocols had an effect on community profile accuracy (Fig. 4A–B), but the largest contributor to variability was contamination or omissions owing to microbes whose DNA was not extracted”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Results > Positive controls
Load-bearingStrength:plain· not yet calibratedCausal:direct· not yet calibrated
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes
It may state two separate claims as one.
- Rests on an assumption: Negative control buffer aliquots are expected to contain no microbial DNA or OTUs, so any OTUs detected in them indicate contamination.
In the paper
“Sources of contamination varied, including one lab with samples comprising mainly Lactobacillus (HL-A), three labs with Pseudomonas (possibly reagent contamination23 , HL-B, HL-F, and HL-D), and sequence, bioinformatic, and/or physical contamination from gut-derived samples in all laboratories.”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Results > Negative controls
What the sources say
- Not checked yetcites [23] Salter, S.J. et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12, 87 (2014). source
Load-bearingStrength:hedged· not yet calibratedCausal:conditional· not yet calibratedn = 345
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes
It may state two separate claims as one.
- Rests on an assumption: Negative control buffer aliquots are expected to contain no microbial DNA or OTUs, so any OTUs detected in them indicate contamination.
In the paper
“Centrally extracted (pre-extracted) samples were associated with higher abundances of Bacteroidetes; since all pre-extracted samples were extracted using a MO-BIO kit, these results are consistent with reports showing that the MO-BIO based HMP protocol generated data enriched for genera within the Bacteroidetes24.”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Results > Multivariate quantification of variation
What the sources say
- Not checked yetcites [24] Wesolowska-Andersen, A. et al. Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis. Microbiome 2, 19 (2014). source
Strength:plain· not yet calibratedCausal:correlational· not yet calibrated
Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passes
It may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“choices of 16S rRNA gene variable region primers may enable improved detection or differentiation of microbes16; for example, Propionibacterium spp. abundant on the skin are often undetected by V4 primers29, and Lactobacillus spp. in the vagina are better differentiated by including V321.”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Discussion
What the sources say
- Not checked yetcites [16] Soergel, D.A., Dey, N., Knight, R. & Brenner, S.E. Selection of primers for optimal taxonomic classification of environmental 16S rRNA gene sequences. ISME J. 6, 1440–1444 (2012). source
- Not checked yetcites [29] Nelson, M.C., Morrison, H.G., Benjamino, J., Grim, S.L. & Graf, J. Analysis, optimization and verification of Illumina-generated 16S rRNA gene amplicon surveys. PLoS One 9, e94249 (2014). source
- Not checked yetcites [21] Fadrosh, D.W. et al. An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome 2, 6 (2014). source
Strength:hedged· not yet calibratedCausal:conditional· not yet calibrated
Tests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passes
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“different human host cohorts may harbor remarkably different Gram-negative versus Gram-positive diversity3, 30, and a protocol appropriate for one type of community may work well in one study but not in another.”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Discussion
What the sources say
- Not checked yetcites [3] Yatsunenko, T. et al. Human gut microbiome viewed across age and geography. Nature 486, 222–227 (2012). source
- Not checked yetcites [30] De Filippo, C. et al. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc. Natl. Acad. Sci. USA 107, 14691–14696 (2010). source
Strength:hedged· not yet calibratedCausal:conditional· not yet calibrated
Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passes
It may state two separate claims as one.
In the paper
“Artificial communities were developed from in-house isolates representative of the range of bacterial genera found within the oral or gut habitats, including both Gram positive and Gram negative bacteria with a range of genomic G+C ratios (Supplementary Table 1).”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Online Methods > Biological specimens
What the sources say
- Not checked yetcites Supplementary Table 1
Load-bearingStrength:plain· not yet calibrated
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes
Probably not a substantial claim.
In the paper
“All strains were originally isolated from human subjects, and HMP genome reference strains were included where available5.”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Online Methods > Biological specimens
What the sources say
- Not checked yetcites [5] Vatanen, T. et al. Variation in microbiome LPS immunogenicity contributes to autoimmunity in humans. Cell 165, 842–853 (2016). source
Strength:plain· not yet calibrated
Tests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passes
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
In the paper
“Sequencing data for positive controls diverge in almost every published study28, including ours (Supplementary Dataset 1), which confirms that relative, not absolute, measures are comparable between protocols.”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Discussion
What the sources say
- Not checked yetcites [28] Morgan, J.L., Darling, A.E. & Eisen, J.A. Metagenomic sequencing of an in vitro-simulated microbial community. PLoS One 5, e10209 (2010). source
Load-bearingStrength:boosted· not yet calibratedCausal:direct· not yet calibrated
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes
It may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“newly diagnosed cases with adenocarcinoma of the colon or rectum were recruited prior to surgery and treatment during 1985–19891, 2. Controls were patients awaiting elective surgery for non-oncologic, non-gastrointestinal conditions at these hospitals during the same period.”
§ Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium > Online Methods > Biological specimens
What the sources say
- Not checked yetcites [1] Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 486, 207–214 (2012). source
- Not checked yetcites [2] Qin, J. et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464, 59–65 (2010). source
Strength:plain· not yet calibratedn = 4
Tests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passes
Probably not a substantial claim. The paper’s conclusions may not depend on it.

87 of 87

The paper relies on this without support.
Negative control buffer aliquots are expected to contain no microbial DNA or OTUs, so any OTUs detected in them indicate contamination.
“We included Tris-HCl buffer aliquots as negative control samples (two per 96-sample set)”
“At least one OTU was reported in 342 of these samples”
The paper relies on this without support.
Comparing centrally extracted DNA with locally extracted DNA isolates the effect of DNA extraction from PCR and sequencing effects.
“We separated variation caused by DNA extraction from variation owing to PCR and sequencing by comparing centrally- versus locally-extracted samples”

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The stated slurry volume exceeds the volume prepared
Major
The procedure prepares a slurry using 50 mL of medium, then says 100 mL of supernatant from the resulting slurry was added to each vessel. No additional slurry preparation is described.
The modeled phylum is named inconsistently
Minor
The results list Bacteroides among the four phyla modeled, whereas the methods list Bacteroidetes in its place. These names identify different taxonomic ranks.
Six laboratories are incorrectly described as a majority
Minor
The paper identifies fifteen handling laboratories, so six laboratories constitute 40%, not a majority.

3 of 3

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Authors: The Microbiome Quality Control Project Consortium, Sinha, R., Abu-Ali, G., Vogtmann, E., Fodor, A. A., Ren, B., Amir, A., Schwager, E., Crabtree, J., Ma, S., Abnet, C. C., Knight, R., White, O., & Huttenhower, C.
Affiliations: National Cancer Institute (US)
Broad Institute (US)
University of North Carolina at Charlotte (US)
Harvard University (US)
University of California San Diego (US)
University of Maryland, Baltimore (US)
Corresponding author: Huttenhower, Curtis
ORCID: Sinha, Rashmi
Abu-Ali, Galeb
Vogtmann, Emily
Fodor, Anthony A
Ren, Boyu
Amir, Amnon
Schwager, Emma
Crabtree, Jonathan
Ma, Siyuan
Abnet, Christian C
Knight, Rob
White, Owen
Huttenhower, Curtis
Venue: Nature Biotechnology
Publisher: Springer Science and Business Media LLC
Published: 2017-10-02
Volume: 35
Issue: 11
Pages: 1077-1086
ISSN: 0733-222X (ISSN-L)
1087-0156
1546-1696
DOI: 10.1038/nbt.3981
PMC: PMC5839636
OpenAlex: W2762425175
Semantic Scholar: a253c43fc33ed458f6d9051ba718eb95e6e7ba1a
Cited by: 587
References: 64
Citations by year: 2026: 104 · 2025: 50 · 2024: 56 · 2023: 60 · 2022: 66 · 2021: 84 · 2020: 60 · 2019: 59 · 2018: 44 · 2017: 3
FWCI: 18.45
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Licence: www.springer.com/tdm (tdm)
Locations: Nature Biotechnology (publishedVersion)
PubMed (publishedVersion)
PubMed Central (acceptedVersion)
Digital Access to Scholarship at Harvard (DASH) (Harvard University) (acceptedVersion)
eScholarship (California Digital Library) (submittedVersion)
Funders: National Human Genome Research Institute (U01HG004866, R01HG005969, U01HG006537, R01HG004872, R01HG005220)
National Cancer Institute (R01CA179243)
National Institute of Diabetes and Digestive and Kidney Diseases (P30DK043351)
National Institute of Dental and Craniofacial Research (U54DE023798)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
Keywords: human gut microbiome, taxonomic profiling, chemostat, DNA extraction, sample handling, bioinformatics protocols, meta-analysis, experimental design
MeSH terms: Gastrointestinal Microbiome, Bacteria, DNA, Bacterial, Humans, Reference Standards, Research Design, Nucleic Acid Amplification Techniques, Microbiota

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Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium

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