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bioconductor-dada2

The dada2 package infers exact amplicon sequence variants (ASVs) from high-throughput amplicon sequencing data, replacing the coarser and less accurate OTU clustering approach. The dada2 pipeline takes as input demultiplexed fastq files, an

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dada2

Dependencies & Environment

Package-intrinsic requirements from the Bioconductor landing page — reproduce in any R environment.

  • Version: 1.40.0 · Bioconductor: 3.23 · R: ≥ 4.6
  • Depends: Rcpp
  • Imports: Biostrings, ggplot2, reshape2, ShortRead, RcppParallel, IRanges, XVector, BiocGenerics
  • System requirements: GNU make
  • Install: BiocManager::install("dada2")

When to Use

  • Resolving exact Amplicon Sequence Variants (ASVs) from high-throughput amplicon sequencing data using the dada algorithm.
  • Filtering and trimming raw paired-end or single-end FASTQ files using filterAndTrim.
  • Modeling and learning sequencing error rates directly from the data using learnErrors.
  • Removing chimeric sequences from merged sequence tables using removeBimeraDenovo.

When NOT to Use

  • For shotgun metagenomic sequencing data (use MetaPhlAn or HUMAnN instead).
  • For clustering sequences based on a fixed 97% identity threshold (use vsearch or UCLUST instead).
  • For downstream ecological statistics and visualization of ASV tables (use phyloseq instead).

Data Requirements

  • Input Format: Demultiplexed FASTQ files (unzipped or gzipped). One file per sample for single-end, or two files per sample for paired-end.
  • Quality Scores: Raw reads must contain quality scores.
  • Primers: Sequences must have non-biological bases (like PCR primers) removed prior to ASV inference.

Key Parameters

  • truncLen: Truncate reads after this many bases. Reads shorter than this are discarded. Crucial for removing low-quality tails.
  • maxEE: After truncation, reads with higher than this number of expected errors are discarded (e.g., maxEE=2).
  • trimLeft: The number of nucleotides to remove from the start of each read (useful for removing primers).
  • maxN: Maximum number of ambiguous nucleotides allowed. DADA2 requires maxN=0.
  • multithread: If TRUE, enables parallel processing to speed up computation.
  • err: The error model generated by learnErrors, passed into the dada function.

Best Practices

  • Inspect the quality profiles of forward and reverse reads using plotQualityProfile before setting truncLen parameters.
  • Learn error rates using learnErrors on at least a subset of your data to ensure accurate sample inference.
  • Maintain a suitable overlap (>20nts) between forward and reverse reads after trimming to ensure mergePairs succeeds.
  • Perform chimera removal using removeBimeraDenovo on the combined sequence table created by makeSequenceTable.

Common Pitfalls

  • Failing to merge paired reads: Truncating reverse reads too aggressively can prevent them from overlapping with forward reads, causing mergePairs to fail. Fix this by ensuring truncLen leaves enough sequence for a >20nt overlap.
  • Leaving primers in the sequences: Non-biological nucleotides distort the error model. Fix this by using trimLeft in filterAndTrim or an external tool to remove primers.
  • Reads dropped due to Ns: DADA2 cannot process sequences with ambiguous bases. Fix this by ensuring maxN=0 is set in filterAndTrim.

Alternatives

  • phyloseq: Used for downstream analysis, visualization, and integration of the ASV tables produced by DADA2.
  • vsearch: An alternative for traditional OTU clustering based on sequence identity thresholds.
  • deblur: An alternative sub-operational taxonomic unit (sOTU) algorithm that uses a static error model.

Citations

  • Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP. (2016). "DADA2: High-resolution sample inference from amplicon data." Nature Methods, 13(7), 581-583.
  • Callahan BJ, McMurdie PJ, Holmes SP. (2017). "Exact sequence variants should replace operational taxonomic units in marker-gene data analysis." ISME Journal, 11(12), 2639-2643.

References

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