NOTE: Use the NextFlow: ONTprocessing pipeline instead of this guide to process Oxford Nanopore data collected for viruses (i.e., insects).
Source:
https://github.com/PrestonLeung/Nano-Q
Create a conda environment:
Prepare an “environment.yml” file that contains the following tool specifications
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name: nanoQ channels: - defaults - anaconda - bioconda - conda-forge dependencies: - python=3.7 - pysam=0.19.1 - numpy=1.23.3 - scipy=1.9.1 - biophytonbiopython=1.79 - matplotlib=3.6.0 - samtools - minimap2 - graphmap - emboss |
Run the following command to generate the ‘nanoQ’ conda environment:
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#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:
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- 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:
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#!/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
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#use a tool provided by emboss
infoseq $REF -only -name -length | sed 1d > ${REF}_list.txt |
submit the job to the scheduler
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qsub launch_variant_analysis.pbs |
Monitor progress
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qjobs |
working example:
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/work/phylo/OxfordNanopore/nextflow/variants/nano-Q/run2 |
Additional information
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#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
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minimap2 -ax asm5 ref.fa asm.fa > aln.sam # assembly to assembly/ref alignment |
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