Description

This tool can be used to remove chimeric sequences, i.e. sequences that can be constructed by combining a left-segment and a right-segment from two more abundant “parent” sequences.. Two methods to identify chimeras are supported: Identification from pooled sequences and identification by consensus across samples.

• from pooled sequences: Each sequence is evaluated against a set of "parents" drawn from the sequence collection that are sufficiently more abundant than the sequence being evaluated. Sequences that are bimera are removed, i.e. a two-parent chimera, in which the left side is made up of one parent sequence, and the right-side made up of a second parent sequence.
• by consensus: In short, bimeric sequences are flagged on a sample-by-sample basis. Then, a vote is performed for each sequence across all samples in which it appeared. If the sequence is flagged in a sufficiently high fraction of samples, it is identified as a bimera. A logical vector is returned, with an entry for each sequence in the table indicating whether it was identified as bimeric by this consensus procedure.

Note: pooled should only be used in combination with pooled denoising.

Usage

Input

• the results of makeSequenceTable (note that also the results of dada, and mergePairs are accepted)

Output

A data set of type: - dada2_sequenceTable (resp. dada2_mergepairs) if the input is of type dada2_sequenceTable (resp. dada2_mergepairs) - dada2_uniques otherwise

Details

The frequency of chimeric sequences varies substantially from dataset to dataset, and depends on on factors including experimental procedures and sample complexity. Here chimeras make up about 21% of the merged sequence variants, but when we account for the abundances of those variants we see they account for only about 4% of the merged sequence reads.

Considerations for your own data: Most of your reads should remain after chimera removal (it is not uncommon for a majority of sequence variants to be removed though). If most of your reads were removed as chimeric, upstream processing may need to be revisited. In almost all cases this is caused by primer sequences with ambiguous nucleotides that were not removed prior to beginning the DADA2 pipeline. You can check for present primer sequences with the tool dada2: primer check

Overview

The intended use of the dada2 tools for paired sequencing data is shown in the following image.

Note: In particular for the analysis of paired collections the collections should be sorted lexicographical before the analysis.

For single end data you the steps "Unzip collection" and "mergePairs" are not necessary.

More information may be found on the dada2 homepage:: https://benjjneb.github.io/dada2/index.html (in particular tutorials) or the documentation of dada2's R package https://bioconductor.org/packages/release/bioc/html/dada2.html (in particular the pdf which contains the full documentation of all parameters)