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Deprecated - ONT variant analysis

Deprecated - ONT variant analysis

Owned by Roberto Barrero Gumiel
Last updated: Aug 09, 2023
2 min read

NOTE: Use the nf-eresearch/ONTprocessing - NextFlow pipeline for Oxford Nanopore de novo assembly and ref guided consensus instead of this guide to process Oxford Nanopore data collected for viruses (i.e., insects).

Source:

GitHub - PrestonLeung/Nano-Q: Python script for conservatively cleaning ONT reads from bam files and estimate variant frequencies.

Create a conda environment:

Prepare an “environment.yml” file that contains the following tool specifications

name: nanoQ channels: - defaults - anaconda - bioconda - conda-forge dependencies: - python=3.7 - pysam=0.19.1 - numpy=1.23.3 - scipy=1.9.1 - biopython=1.79 - matplotlib=3.6.0 - samtools - minimap2 - graphmap - emboss

Run the following command to generate the ‘nanoQ’ conda environment:

conda env create -f environment.yml

Activate the environment to access the installed tools:

conda activate nanoQ

Alternatively - manually run each of the following commands:

#create an interactive session qsub -I -S /bin/bash -l walltime=10:00:00 -l select=1:ncpus=2:mem=4gb #create conda environment conda create --name nanoQ2 python=3.7 conda activate nanoQ2 #install tools conda install -c bioconda pysam conda install -c conda-forge numpy conda install -c conda-forge scipy conda install -c conda-forge matplotlib conda install -c conda-forge biopython conda install -c bioconda samtools #aligners conda install -c bioconda minimap2 conda install -c bioconda graphmap #fetch nano-Q git clone https://github.com/PrestonLeung/Nano-Q.git #change directory to Nano-Q cd Nano-Q #make all python scripts executable chmod +x *.py #copy all python scripts to your /home/bin/ cp *.py ~/bin

Map long ONT reads onto a reference genome

Input:

- ref_genome.fasta - ref_list.txt # length of sequences in the above file: Accession "tab" Length (i.e., NC001477 10735) - sample FASTQ file(s)

Prepare a script, for example, called ‘launch_variant_analysis.pbs’ that contains the following information:

#!/bin/bash -l #PBS -N nano-Q #PBS -l walltime=24:00:00 #PBS -l mem=16gb #PBS -l ncpus=8 cd $PBS_O_WORKDIR ################################################################################################################################ # USER DEFINE VARIABLES ################################################################################################################################ SAMPLEID=NC483 REFNAME=NC001477_RefGenome LIST='/myteam/OxfordNanopore/nextflow/assembly/data/NC483/NC483_NC001477_ref_list.txt' REF='/myteam/OxfordNanopore/nextflow/assembly/data/NC483/NC483_NC001477_reference_sequence.fasta' ONT='/myteam/OxfordNanopore/nextflow/assembly/data/NC483/NC483_FAU10290_pass_barcode96_0cf303ee.fastq' ################################################################################################################################ #activate conda environment containing tools for analysis conda activate nanoQ2 #generic mapping reads minimap2 -a $REF $ONT > ${SAMPLEID}_aln.sam #mapping noisy reads #minimap2 -ax $REF $ONT > ${SAMPLEID}_aln2.sam #Samtools samtools view -bt ${LIST} -o ${SAMPLEID}_aln.bam ${SAMPLEID}_aln.sam samtools sort -T /tmp/aln.sorted -o ${SAMPLEID}_aln.sorted.bam ${SAMPLEID}_aln.bam samtools index ${SAMPLEID}_aln.sorted.bam #run nano-Q python /work/phylo/OxfordNanopore/nextflow/tools/git/Nano-Q/nano-q.py -b ${SAMPLEID}_aln.sorted.bam -c 1 -l 9000 -nr 1 -q 5 -j 10

creating a ref_list.txt file

#use a tool provided by emboss infoseq $REF -only -name -length | sed 1d > ${REF}_list.txt

 

submit the job to the scheduler

qsub launch_variant_analysis.pbs

Monitor progress

qjobs

working example:

/work/phylo/OxfordNanopore/nextflow/variants/nano-Q/run2

 

Additional information

#generic mapping reads minimap2 -a ref.fa ont-reads.fq > aln.sam #mapping noisy reads minimap2 -ax map-ont ref.fa ont-reads.fq > aln.sam # for Oxford Nanopore reads

Full genome/assembly alignment

minimap2 -ax asm5 ref.fa asm.fa > aln.sam # assembly to assembly/ref alignment

 

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