DADA2: High–resolution sample inference from Illumina amplicon data
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Abstract
We present the open-source software package DADA2 for modeling and correcting Illumina-sequenced amplicon errors (https://github.com/benjjneb/dada2). DADA2 infers sample sequences exactly and resolves differences of as little as 1 nucleotide. In several mock communities, DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants.
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Quality control, exact sequence variants clustering, and chimera removal for 16S sequences were conducted using DADA2.
“Quality control, exact sequence variants clustering, and chimera removal were conducted using the dada2 package76 for R (R Core Team 2017).”
From Integrated analysis of behavioral, epigenetic, and gut microbiome analyses in AppNL-G-F, AppNL-F, and wild type mice (2021) · cites this paper as [76]
Methods for amplicon-based community profiling have progressed from noise- and chimera-prone stitching and clustering to near-exact sequence variant tracking.
“Similarly, methods for amplicon-based community profiling have progressed from noise- and chimera-prone stitching and clustering to near-exact sequence variant tracking (Callahan et al., 2016).”
From Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3 (2021) · cites this paper as Callahan et al., 2016
The V3-V4 region sequencing resulted in 24,952,554 total reads; after DADA2 filtering, 16,813,759 reads with average 70,350 ± 57,098 reads per sample, resulting in 14,839 ASVs.
“The V3-V4 region of 16S rDNA sequencing of the gut microbiota resulted in a total 24.952.554 reads. After DADA2 filtering step, we obtained a total of 16.813.759 reads with an average of 70.350 ± 57.098 reads for each sample, resulting in 14.839 ASVs [39].”
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- Authors
- Callahan, B. J., McMurdie, P. J., Rosen, M. J., Han, A. W., Johnson, A. J. A., & Holmes, S. P.
- Affiliations
- Stanford University (US)
- Second Genome (United States) (US)
- Corresponding author
- Callahan, Benjamin J
- Venue
- Nature Methods
- Published
- 2016-05-23
- Volume
- 13
- Issue
- 7
- Pages
- 581-583
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC4927377
- OpenAlex
- W2401404581
- Cited by
- 35,698
- References
- 28
- Citations by year
- 2026: 5430 · 2025: 6295 · 2024: 6207 · 2023: 5211 · 2022: 4649 · 2021: 3876 · 2020: 2419 · 2019: 1043 · 2018: 418 · 2017: 101 · 2016: 21 · 2015: 2 · 2013: 2 · 2012: 1
- FWCI
- 272.82
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- cc-by-nc-nd
- www.springer.com/tdm (tdm)
- Locations
- Nature Methods (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Science Foundation (1162538)
- National Institutes of Health
- National Institute of Allergy and Infectious Diseases (R01AI112401)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
- Keywords
- DADA2, amplicon sequence variants, sequencing error correction, noise reduction, microbial community sequencing, single-nucleotide resolution, mock communities, vaginal microbiota, Lactobacillus crispatus, pregnant women, open-source bioinformatics software
- MeSH terms
- Animals, DNA, Bacterial, False Positive Reactions, Feces, Female, Humans, Lactobacillus, Pregnancy, RNA, Ribosomal, 16S, Software, Vagina, Reproducibility of Results, Cohort Studies, Sequence Analysis, DNA, Computational Biology, Mice, High-Throughput Nucleotide Sequencing, Microbiota
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