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Table of Contents
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Aims
Implement a bioinformatics pipeline for the detection of EBV-integration sites in the human genome
Generate ONT simulated data
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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.
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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:
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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:
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cp slow5tools $HOME/bin/ |
Example data:
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/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:
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./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:
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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:
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#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} |