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ONT de novo genome assembly

ONT de novo genome assembly

Owned by Roberto Barrero Gumiel
Last updated: Oct 06, 2022
2 min read

Aim:

Implement a de novo assembly pipeline for ONT data

Source:

https://nanoporetech.com/sites/default/files/s3/literature/Bacterial-assembly-workflow.pdf

https://github.com/fenderglass/Flye/blob/flye/docs/INSTALL.md

Methodology

a) create a conda environment with Flye and its dependencies

Prepare a file called ‘environment.yml’ that contains the following information:

name: flye channels: - defaults - anaconda - bioconda - conda-forge dependencies: - python=3.7 - flye=2.9.1 - seqkit=2.3.1 - blast=2.13.0 - nanoq=0.9.0 - minimap2 - samtools

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

conda env create -f environment.yml

Activate the environment to access the installed tools:

conda activate flye

merge FASTQ files

cat FAU10290_pass_barcode96_0cf303ee_*.gz > merge_NC483.fastq.gz

get path of merged file

pwd

modify launch*.pbs file

 

b) Run a de novo assembly and sequence comparison against a Reference genome

#!/bin/bash -l #PBS -N FlyeAssembly #PBS -l walltime=24:00:00 #PBS -l mem=16gb #PBS -l ncpus=8 ## Usage: qsub launch_flye_genome_assembly.pbs cd $PBS_O_WORKDIR ################################################################################################################################ # USER DEFINED VARIABLES ################################################################################################################################ SAMPLEID=MyGenome REFNAME=Accession_RefGenome REF='/path/to/REF/genome.fasta' ONT='/path/to/ONT/reads.fastq.gz' ################################################################################################################################ #activate flye conda environment conda activate flye #ASSEMBLY #STEP1: Run de novo genome assembly for >=Q20 data, use a combination of --nano-hq and --read-error 0.03 flye --out-dir outdir_nano --threads 8 --read-error 0.03 --nano-hq $ONT #STEP2: Compare sequence similarity of assembled genome with reference sequence blastn -query outdir_nano/assembly.fasta -subject $REF -evalue 1e-5 -out blastn_${REFNAME}_vs_${SAMPLEID}_assembly.txt -outfmt '6 qseqid sacc length pident mismatch gapopen qstart qend qlen sstart send slen evalue bitscore qcovhsp qcovs' #STEP3: format output BLASTN table echo "qseqid sacc length pident mismatch gapopen qstart qend qlen sstart send slen evalue bitscore qcovhsp qcovs" > header #add header to blast output cat header blastn_${REFNAME}_vs_${SAMPLEID}_assembly.txt > BLASTN_${REFNAME}_vs_${SAMPLEID}_assembly.txt #remove intermediate files rm header blastn_${REFNAME}_vs_${SAMPLEID}_assembly.txt #MAPPING #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

submit the job

qsub launch_flye_genome_assembly.pbs

 

monitor progress of the assembly:

qjobs

 

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