cmuts align

Purpose

cmuts align is a simple wrapper script for performing trimming, demultiplexing, and alignment of one or more FASTQ/BAM files against a reference FASTA file, and sorting of the resulting SAM files. This is required if running cmuts directly from raw sequencing data.

Usage

Alignment

By default, cmuts align will perform alignment only, which also includes building the bowtie2 index files and sorting the output SAM files. The syntax is

cmuts align \
  --fasta "$FASTA" \
  --threads "$THREADS" \
  --output "$ALIGNMENTS" \
  "$FASTQ"/*.fastq*

The sorted alignments will be in BAM format and can be found in the directory $ALIGNMENTS, with the same basename as the original FASTQ files.

The default is for cmuts align to use --end-to-end alignment. To use --local alignment instead, simply pass the --local argument.

Paired-End Alignment

To specify mates for paired-end sequencing, pass the additional FASTQ files to the --pairs argument after the forward reads.

cmuts align \
  --fasta "$FASTA" \
  --threads "$THREADS" \
  --output "$ALIGNMENTS" \
  "$FASTQ"/*.fastq* \
  --pairs "$PAIRS"/*.fastq*

Warning

The --pairs inputs must come after the forward reads, otherwise cmuts align will not be able to distinguish what you intend to be the mate.

Demultiplexing

Pre-alignment demultiplexing (via ultraplex) can be achieved by passing a CSV of barcodes to the --barcodes argument.

cmuts align \
  --fasta "$FASTA" \
  --threads "$THREADS" \
  --barcodes "$BARCODES" \
  --output "$ALIGNMENTS" \
  "$FASTQ"/*.fastq*

The sorted alignments will again be in BAM format under the directory $ALIGNMENTS. However, their names will be given by the respective barcode which they correspond to, not the name of the original FASTQ file.

Warning

If the barcodes are named in the CSV file, then the outputs will have those as names rather than the respective barcodes (see ultraplex documentation).

Trimming

Pre-alignment trimming (via cutadapt) can be achieved by passing a sequence to either the --trim-5 or --trim-3 argument, depending on which side the adapter belongs to. Separate adapters can be passed for each.

cmuts align \
  --fasta "$FASTA" \
  --threads "$THREADS" \
  --trim-5 "$TRIM5" \
  --trim-3 "$TRIM3" \
  --output "$ALIGNMENTS" \
  "$FASTQ"/*.fastq*

Trimming does not change the format of the output.

Warning

If at least one of --trim-5 and --trim-3 are passed with --barcodes specified, the trimming will occur first.

Other Arguments

--overwrite : Overwrite the output directories, including any temporary directories used for demultiplexing and trimming.

--log : Specify the log file to use. Default: cmuts-align.log