kromsatel

Current version is 2.0.b (2022-02-23 edition).

Kromsatel is a program which preprocesses ("cleans") raw reads of amplicon sequencing.

For example, when the genome of SARS-CoV-2 is sequenced, raw reads of amplicons produced using ARTIC protocol can be processed with kromsatel before any downstream sequence analysis. Any other amplicon protocol is (most likely) also acceptable; however, only ARTIC has been tested.

Description

Acceptable input data

• short reads (e.g. Illumina). Reads may be either paired-end or single-end. Kromsatel was tested on Illumina data.

• long reads (e.g. Oxford Nanopore). Kromsatel was tested on Oxford Nanopore data.

Preprocessing: specifics

For kromsatel, the "preprocessing" stands for the following:

1. Remove primer sequences from reads.

2. Trim reads so that any unaligned part of read is removed.

3. Discriminate reads coming from major, minor, and non-specific amplicons (see the picture below, TODO).

4. Split chimeric reads into consistent fragments (only long reads).

Dependencies

1. Python 3 (https://www.python.org/). The script is tested on Python interpreter version 3.8.5.

The script is written in Python 3 and won't work on Python 2.

2. BLAST+ toolkit.

   It can be downloaded here.

   BLAST+ Installation:

   • Linux: download tarball, unpack it and add bin/ directory from unpacked tree to PATH variable. (this should also work for macOS)

   Kromsatel has been tested on Linux with BLAST+ version 2.12.0+.

Usage

Arguments

Mandatory arguments are marked with *.

  -h (--help) -- print the help message and exit.

  -v (--version) -- print version and exit.

Input:

  -1 (--reads-R1) -- a fastq file of forward reads.
      The file may be gzipped.

  -2 (--reads-R2) -- a fastq file of reverse reads.
      The file may be gzipped.

  -l (--reads-long) -- a fastq file of long reads.
      The file may be gzipped.

* -p (--primers) -- a CSV file of primer names and sequences.
      This file must be a two-column CSV file without header.

* -r (--reference) -- a fasta file of reference sequence.

Output:

  -o (--outdir) -- output directory.
      Default value is './kromsatel_output'

  -s (--split-output) -- split ouput files into 3 "classes":
      "major", "minor" and "uncertain".
      Disabled by default.

Computational resources:

  -t (--threads) -- number of threads to launch.
      Default: 1 thread.

Advanced:

  -m (--min-len) -- minimum length of an output read.
      Default: 25 bp.

  -k (--blast-task) -- BLASTn task to launch.
      Allowed values: 'megablast', 'dc-megablast', 'blastn'.
      Default is 'megablast'.

  -c (--chunk-size) -- number of reads to blast within a single query.
      The larger is the chunk size, the higher is the memory consumption.
      Default: 1000 reads.

  --crop-len -- number of nucleotides to crop from end of reads
      originating from a non-specific amplicon.
      Default: 'auto' (maximum primer length).

  --primer-5ext -- size of 5' primer coordinates extention.
      Low (< 2 bp) values of this parameter may result in extra major alignments
      classified as uncertain, and vice versa for high values (> 10 bp).
      Default: 5 bp.

  --use-index -- Whether to use BLAST index.
      Permitted values: auto, true, false.
      "auto" mode: true for megablast and blastn, false for dc-megablast.
      Default: false.

Examples

Short (e.g. Illumina) paired-end reads

./kromsatel.py \
    -1 20_S30_L001_R1_001.fastq.gz \
    -2 20_S30_L001_R2_001.fastq.gz \
    -p primers/nCov-2019_primers.csv \
    -r reference/Wuhan-Hu-1-compele-genome.fasta \
    -o 20_S30_outdir

Short (e.g. Illumina) single-end reads

./kromsatel.py \
    -1 20_S30_L001_R1_001.fastq.gz \
    -p primers/nCov-2019_primers.csv \
    -r reference/Wuhan-Hu-1-compele-genome.fasta \
    -o 20_S30_outdir

Long (e.g. Oxford Nanopore) reads

./kromsatel.py \
    -l all_pass_15.fastq.gz \
    -p primers/nCov-2019_primers.csv \
    -r reference/Wuhan-Hu-1-compele-genome.fasta \
    -o Wuhan-Hu-1_outdir

With additional options

./kromsatel.py \
    -1 20_S30_L001_R1_001.fastq.gz \
    -2 20_S30_L001_R2_001.fastq.gz \
    -p primers/nCov-2019_primers.csv \
    -r reference/Wuhan-Hu-1-compele-genome.fasta \
    -o 20_S30_outdir \
    -k dc-megablast \
    -c 2000 \
    -m 50 \
    -t 4 \
    --crop-len 27 \
    --primer-5ext 3

Output read names

Short (e.g. Illumina) reads

Kromsatel keeps read headers unchanged.

Long (e.g. Nanopore) reads

For Nanopore data, kromsatel modifies headers of output reads. Thus, the header of an output read will looklike this:

  @<ORIGINAL_READ_NAME>_<QSTART>-<QEND> [runid,sampleid,etc...]

, where QSTART and QEND are 1-based coordinates of, correspondingly, start and end of the aligned fragment, which yields the output read.

An example of a modified read name in an output file:

  @98786bd2-88a6-43ca-8c69-704992ad69cb_28-98 runid=...,sampleid=...,etc...