Search and remove primers and sample indexes

do_trim_demux is a wrapper function running do_primer_search and do_demux sequentially.

Usage

do_trim_demux(
  fq_paths,
  amplicon_primers,
  sample_tab,
  out_dir,
  primer_max_err = 0.2,
  idx_max_diffs = 0,
  min_barcode_length = 50,
  error_threshold = 2.5,
  cores = NULL,
  keep_trimmed = FALSE
)

Arguments

fq_paths

Character vector of FASTQ file paths (compressed or uncompressed)

amplicon_primers

Amplicon specification, as returned by parse_primer_tab

sample_tab

Sample table as returned by parse_sample_tab

out_dir

Output directory containing the demultiplexed files (fprimer_fidx-rprimer_ridx.fastq.gz)

primer_max_err

Maximum allowed error rate in primers (edit distance = substitutions/InDels). Default: 0.2 (20%)

idx_max_diffs

Maximum allowed differences in forward/reverse sample index sequences; don't set too high to avoid tag switching ("index hopping") resulting in a "background noise" of unspecific sequences. Default: 0 differences

min_barcode_length

Remove trimmed sequences shorter than min_barcode_length; make sure that the value is shorter than your expected minimal barcode length for all amplicons (default: 50 bp)

error_threshold

Maximum sequencing errors allowed per sequence (as estimated from quality scores). This threshold impacts how many sequences pass the filter in (see do_demux). Setting it too low may remove too many sequences, but setting it too high may lead to noisy/errorneous results. The default value of 2.5 seems to work well with R10.4 data and ~0.5-1.5 kb amplicons, removing about half of the trimmed sequences. The value may still be adjusted based on how Figures 4 and 5 in the HTML report.

cores

Number of cores to use (default: 1, unless getOption('DadaNanoBC.cores') is defined, or the DadaNanoBC_cores environment variable is set)

Value

A list of:

• seq_tab: sample_tab with a reads_path column and additional rows for samples found in the reads but not in the sample table

• trim_stats: a list containing primer/index search statistics (used in the HTML report)

Details

Requires seqtool v0.4 or higher installed either system-wide (in PATH) or locally (provide path with set_program_path('seqtool', 'path/to/st')).