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EBV genome integration data analysis

EBV genome integration data analysis

Owned by Roberto Barrero Gumiel
Last updated: Apr 22, 2024
3 min read

Table of Contents

Aims

  • Implement a bioinformatics pipeline for the detection of EBV-integration sites in the human genome

Generate ONT simulated data

squigulator is a tool for simulating nanopore raw signal data. It is under development and there could be interface changes and changes to default parameters. Read more here: GitHub - hasindu2008/squigulator: a tool for simulating nanopore raw signal data

Install a precompiled copy:

VERSION=0.3.0 wget https://github.com/hasindu2008/squigulator/releases/download/v${VERSION}/squigulator-v${VERSION}-x86_64-linux-binaries.tar.gz tar xf squigulator-v${VERSION}-x86_64-linux-binaries.tar.gz && cd squigulator-v${VERSION} ./squigulator --help

Location: /work/GRC_collaborations/EBV/tools/squigulator-v0.3.0

PHASE 1: No mutations introduced to reference genomes prior simulation of ONT data.

Genomes:

  • GRCh38.p14

  • EBV_ASM240226v1

  • GRCh38+EBV_ASM240226v1 (integrated in the genome)

GRCh38.p14 ONT simulated data:

Location:

/work/GRC_collaborations/EBV/analysis/1.squigulator_simulated_data_NoVariants/run1_GRCh38.p14_human_genome

Squigulator simulation:

#!/bin/bash -l #PBS -N squigulator_GRCh38.p14 #PBS -l walltime=48:00:00 #PBS -l mem=64gb #PBS -l ncpus=32 #use current working directory cd $PBS_O_WORKDIR ################################################# ## user defined variables ################################################# SQUIGULATOR='/work/GRC_collaborations/EBV/tools/squigulator-v0.3.0/squigulator' SAMPLEID='GRCh38.p14' GENOME='/work/GRC_collaborations/EBV/Genomes/GCF_000001405.40_GRCh38.p14_genomic.fna' COVERAGE=30 PROFILE='dna-r10-prom' ################################################# #STEP1: Create simulated reads at 30X genome coverage #example code: #squigulator hg38noAlt.fa -x dna-r10-prom -o reads.blow5 -f 30 #we use the user defined variables above to modify the example code: $SQUIGULATOR $GENOME -x $PROFILE \ -o ${SAMPLEID}_ONT_${PROFILE}_reads.blow5 \ -f $COVERAGE \ -t 32 \ -q ${SAMPLEID}_ONT_${PROFILE}_reads.fasta \ -c ${SAMPLEID}_ONT_${PROFILE}_reads_aln.paf \ -a ${SAMPLEID}_ONT_${PROFILE}_reads_aln.sam

EBV_ASM240226v1 ONT simulated data:

Location:

/work/GRC_collaborations/EBV/analysis/1.squigulator_simulated_data_NoVariants/run2_ASM240226v1_viral_genome

Squigulator simulation:

#!/bin/bash -l #PBS -N squigulator_EBV #PBS -l walltime=24:00:00 #PBS -l mem=32gb #PBS -l ncpus=16 #use current working directory cd $PBS_O_WORKDIR ################################################# ## user defined variables ################################################# SQUIGULATOR='/work/GRC_collaborations/EBV/tools/squigulator-v0.3.0/squigulator' SAMPLEID='EBV_ASM240226v1' GENOME='/work/GRC_collaborations/EBV/Genomes/GCF_002402265.1_ASM240226v1_genomic.fna' COVERAGE=30 PROFILE='dna-r10-prom' ################################################# #STEP1: Create simulated reads at 30X genome coverage #example code: #squigulator hg38noAlt.fa -x dna-r10-prom -o reads.blow5 -f 30 #we use the user defined variables above to modify the example code: $SQUIGULATOR $GENOME -x $PROFILE \ -o ${SAMPLEID}_ONT_${PROFILE}_reads.blow5 \ -f $COVERAGE \ -t 16 \ -q ${SAMPLEID}_ONT_${PROFILE}_reads.fasta \ -c ${SAMPLEID}_ONT_${PROFILE}_reads_aln.paf \ -a ${SAMPLEID}_ONT_${PROFILE}_reads_aln.sam

GRCh38.p14+EBV_ASM240226v1 ONT simulated data:

Locations:

/work/GRC_collaborations/EBV/analysis/1.squigulator_simulated_data_NoVariants/run3_custom_genome_human+virus

Squigulator simulation:

#!/bin/bash -l #PBS -N squigulator_GRCh38.p14_EBV #PBS -l walltime=48:00:00 #PBS -l mem=64gb #PBS -l ncpus=32 #use current working directory cd $PBS_O_WORKDIR ################################################# ## user defined variables ################################################# SQUIGULATOR='/work/GRC_collaborations/EBV/tools/squigulator-v0.3.0/squigulator' SAMPLEID='GRCh38.p14_EBV_custom_genome' GENOME='/work/GRC_collaborations/EBV/Genomes/custom_genome_one_GCF.fna' COVERAGE=30 PROFILE='dna-r10-prom' ################################################# #STEP1: Create simulated reads at 30X genome coverage #example code: #squigulator hg38noAlt.fa -x dna-r10-prom -o reads.blow5 -f 30 #we use the user defined variables above to modify the example code: $SQUIGULATOR $GENOME -x $PROFILE \ -o ${SAMPLEID}_ONT_${PROFILE}_reads.blow5 \ -f $COVERAGE \ -t 32 \ -q ${SAMPLEID}_ONT_${PROFILE}_reads.fasta \ -c ${SAMPLEID}_ONT_${PROFILE}_reads_aln.paf \ -a ${SAMPLEID}_ONT_${PROFILE}_reads_aln.sam

Outputs include:

  • BLOW5: simulated ONT data using dna-r10-prom profile at 30X genome coverage (*reads.blow5)

  • FASTA: FASTA file to write simulated reads with no errors (*reads.fasta)

  • PAF: PAF file to write the alignment of simulated reads (*reads_aln.paf)

  • SAM: SAM file to write the alignment of simulated reads (*reads_aln.sam)

Converting BLOW5 to FAST5 data

First, let’s install ‘slow5tools’ from GitHub:

VERSION=v1.1.0 wget "https://github.com/hasindu2008/slow5tools/releases/download/$VERSION/slow5tools-$VERSION-x86_64-linux-binaries.tar.gz" && tar xvf slow5tools-$VERSION-x86_64-linux-binaries.tar.gz && cd slow5tools-$VERSION/ ./slow5tools

Optionally, copy the ‘slow5tools’ executable to your home bin:

cp slow5tools $HOME/bin/

Example data:

/work/GRC_collaborations/EBV/analysis/1.squigulator_simulated_data_NoVariants/coverage_5x/run2_ASM240226v1_viral_genome/EBV_ASM240226v1_ONT_dna-r10-prom_reads_5x.blow5

slow5tools parameters:

./slow5tools --help Usage: ./slow5tools [OPTIONS] [COMMAND] [ARG] Tools for using slow5 files. OPTIONS: -h, --help Display this message and exit. -v, --verbose Verbosity level. -V, --version Output version information and exit. --cite Prints the citation. COMMANDS: f2s or fast5toslow5 convert fast5 file(s) to SLOW5/BLOW5 s2f or slow5tofast5 convert SLOW5/BLOW5 file(s) to fast5 merge merge SLOW5/BLOW5 files split split SLOW5/BLOW5 files index create a SLOW5/BLOW5 index file get display the read entry for each specified read id view view the contents of a SLOW5/BLOW5 file or convert between different SLOW5/BLOW5 formats and compressions stats prints statistics of a SLOW5/BLOW5 file to the stdout cat quickly concatenate SLOW5/BLOW5 files of same type (same header, extension, compression)] quickcheck quickly checks if a SLOW5/BLOW5 file is intact skim skims through requested components in a SLOW5/BLOW5 file ARGS: Try './slow5tools [COMMAND] --help' for more information.

Convert slow5 to fast5 - options:

Convert SLOW5/BLOW5 files to FAST5 format. Usage: ./slow5tools s2f [OPTIONS] -d [OUT_DIR] [SLOW5_FILE/DIR] ... OPTIONS: -d, --out-dir DIR output to directory -o, --output FILE output to FILE [stdout] -p, --iop INT number of I/O processes [8] -h, --help display this message and exit

Running slow5rools:

#PBS -N Convert_EBV #PBS -l walltime=24:00:00 #PBS -l mem=16gb #PBS -l ncpus=8 #PBS -m abe #PBS -o convert_blow5_to_fast5_output.log #PBS -e convert_blow5_to_fast5_error.log #Positive control ###Simulated data: convert BLOW5 to FAST5 for wf-basecalling #### Note: Adjust the path directories # Change to the directory where the job was submitted cd $PBS_O_WORKDIR # Load the necessary modules module load slow5tools # Define path directories as a variable BLOW5_FILE='GRCh38.p14_ONT_dna-r10-prom_reads_5x.blow5' FAST5_FILE='GRCh38.p14_ONT_dna-r10-prom_reads_5x.fast5' SLOW5TOOLS='/work/GRC_collaborations/EBV/tools/slow5tools-v1.1.0/slow5tools' # Ensure the output directory exists # when multiple slow5 files are available then use -d outpur_dir option rather than -o converted.fastq file (see below) #mkdir -p ${OUT_DIR} # Run the conversion #slow5tools tofast5 -o ${FAST5_FILE} ${BLOW5_FILE} $SLOW5TOOLS s2f -p 8 -o ${FAST5_FILE} ${BLOW5_FILE}

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