Long Read Processing Pipeline

A nextflow pipeline of processing long reads

Table of Contents

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1. Dependencies
2. File Format
   • Structure of input directories
   • Sample sheet
3. Usage
   • Run job
   • Usage options

Dependencies

• nextflow
• minimap2
• samtools
• bamtools
• python packages
  • pysam
  • biopython
  • numpy
  • scipy
  • matplotlib
  • pandas

File Format

Structure of input directories

Sample Sheet -- csv

group	barcode_start	barcode_end	barcode_template	directory	reference	gene_info
bc2081	5832	5869	NNNNATNNNNATNNNNATNNNNATNNNNATNNNNATNN	/path/of/directory/	GUK1_PGJJ162.fa	bc2081.txt
bc2082	4819	4856	NNNNATNNNNATNNNNATNNNNATNNNNATNNNNATNN	/path/of/directory/	GUK1_PTB198.fa	bc2082.txt
bc2083	4791	4828	NNNNATNNNNATNNNNATNNNNATNNNNATNNNNATNN	/path/of/directory/	GUK1_PH003.fa	bc2083.txt

Note

1. The sample sheet must be a csv file and the header must be like below in the example
2. For barcode association, you need everything in the field
3. For variant coverer plot, you only need group, directory, reference

Gene Info -- tsv

hGUK1_wt	6256	591
yGUK1_wt	6256	561
pGUK1_wt	6256	597

Note

1. The gene info file must be a tsv file and in the directory according to the sample sheet
2. The gene info file has no header
3. The first column is the reference ID corresponding to the gene (check your reference fasta file)
4. The second column is the start position of the gene in the reference
5. The third column is the length of the gene

Usage

Run job

submit the bash script below

#!/bin/bash
#BSUB -o %J.o
#BSUB -e %J.e
#BSUB -R "select[mem>1000] rusage[mem=1000]"
#BSUB -M 1000
#BSUB -q normal

# modules
module load HGI/common/nextflow/23.10.0
module load HGI/softpack/users/fs18/nf_longread

#--------------#
# user specify #
#--------------#
# LSF group
export LSB_DEFAULT_USERGROUP=hgi

# Paths
export INPUTSAMPLE=$PWD/inputs/samplesheet.csv
export OUTPUTRES=$PWD/outputs

#-----------#
# pipelines #
#-----------#
nextflow run -resume nf_longread/main.nf --sample_sheet $INPUTSAMPLE \
                                         --protocol DNA \
                                         --platform hifi \
                                         --outdir $OUTPUTRES \
                                         --skip_snvcov

Usage options

nextflow run check_inputs.nf --sample_sheet "/path/of/sample/sheet"

    Mandatory arguments:
        --sample_sheet        Path of the sample sheet
    
    Optional arguments:
    Basic:
        --outdir              the directory path of output results, default: the current directory
    
    Alignment:
        --protocol            DNA, cDNA, directRNA, default: DNA
        --platform            nanopore, pacbio, hifi, default: nanopore

    Barcode Detection:
        --mapq                the mapping quality for filtering, default: 1
        --qualcut             the base quality in the barcode for filtering , default: 10
        --numcut              the number of low-quality bases in the barcode for filtering, default: 3
        --countcut            the number of reads supporting the barcode for filtering, default: 5

    Extract SNVs:
        --basequal            the base quality for filtering, default: 30
        --region              the expected region of variants, eg: 100,200, default: 0,0

    Step arguments:
        --skip_align          skip alignment
        --skip_barcode        skip barcode detection
        --skip_snvcov         skip snv coverage extraction