GitHub - griffithlab/htlv_integration_sites: HTLV-1 integration site analysis notes and scripts

Example of an HTLV-1 integration site analysis

Short hand sample names: CTCF-1, CTCF-7, CTCF-8, P12-10B, P12-14, P12-5, P12-8

High level questions

What are the experimental details here? Humanized mice (humanized how?) are infected with different strains of HTLV-1? Four different strains here? And we are looking for genome integrations in mice cells? or human cells? How were the cells obtained for genomic DNA isolation (is this just from blood?). The goal here is to identify the viral integration sites and quantify them to assess clonality? Is there an expectation for degree of clonality we might observe. Are we expecting to see many different unique integration sites in each sample?

Brief answers: Genomic DNA was isolated from humanized mouse spleen that was infected with HTLV-1 p12(wt control) or CTCF mutant virus. p12-10B, p12-14 and CTCF-7, CTCF-8 are mouse ID numbers. We want to quantify the viral integration sites in infected human T cells to assess clonality. I expect to see many unique integration sites but don’t know what kinds of clonality that would be observed.

CD34+ cells were injected in liver at 1d of life. Infected with HTLV. 2 strains – p12 and CTCF. Analysis focused on human cell DNA. Samples were obtained from spleen. The goal here is to identify the viral integration sites and quantify them to assess clonality. Using Gini index value.

Samples in each batch

Batch 1: CTCF-7, CTCF-8, P12-10B,P12-14
Batch 2: CTCF-1, CTCF-3, P12-5, P12-8

Set ENVs

If needed update the following in envs.txt so they can be sourced when needed

export WORKING_DIR=/storage1/fs1/mgriffit/Active/griffithlab/adhoc/ratner_p01/htlv_integration_sites

export FASTQ_NAMES=("Ratner_CTCF-1_SIC_934_196_CGTATCTCAA_AATACTAATA_S156_" "Ratner_CTCF-3_SIC_935_196_GTCCTGCCGA_AATACTAATA_S157_" "Ratner_CTCF-7_SIC_934_SIC2_Ratner_196_CGTATCTCA_AATACTAATA_S2_" "Ratner_CTCF-8_SIC_935_SIC2_Ratner_196_GTCCTGCCG_AATACTAATA_S3_" "Ratner_P12-10B_SIC_936_SIC2_Ratner_196_CCGGGACAC_AATACTAATA_S4_" "Ratner_P12-14_SIC_937_SIC2_Ratner_196_GGCTGGGAT_AATACTAATA_S5_" "Ratner_P12-5_SIC_936_196_CCGGGACACA_AATACTAATA_S158_" "Ratner_P12-8_SIC_937_196_GGCTGGGATA_AATACTAATA_S159_")

export PAIRS=("R1" "R2")

export SEQS=("TTAGTACACA" "AATCATGTGT" "TGACAATGAC" "ACTGTTACTG")

Download the data

sh $WORKING_DIR/git/htlv_integration_sites/scripts/download_raw_data.sh

Get the file base names:

cd $WORKING_DIR
ls -1 fastqs/* | perl -ne 'chomp; if ($_ =~ /(.*)\_\S+\_\S+\.fastq\.gz$/){print "$1_\n"}' | sort | uniq -c 
ls -1 fastqs/*| perl -ne 'chomp; if ($_ =~ /(.*)\_\S+\_\S+\.fastq\.gz$/){print "$1_\n"}' | sort | uniq

Investigate the four supplies possible integration characteristic sequences:

TTAGTACACA / AATCATGTGT TGACAATGAC / ACTGTTACTG

cd $WORKING_DIR
for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
  for PAIR in "${PAIRS[@]}"; do
    for SEQ in "${SEQS[@]}"; do
      ANSWER=$(zcat fastqs/${FASTQ_NAME}${PAIR}_001.fastq.gz | awk 'NR % 4 == 2' | grep $SEQ | wc -l)
      echo "$FASTQ_NAME $PAIR $SEQ $ANSWER"
    done
  done
done

Based on this analysis it seems that for these data in the RAW read sequences we only really see the "TTAGTACACA" sequence and only in Read 1 files

Create unique read lists of these read identities and store them for later use

cd $WORKING_DIR
for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    echo -e "\nProcessing FASTQ: $FASTQ_NAME (R1 only)"
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')
    echo "Will name output using sample name: $SAMPLE"
    zcat fastqs/${FASTQ_NAME}R1_001.fastq.gz | awk 'NR % 4 == 1 {read_name = substr($1, 2)} NR % 4 == 2 {print read_name, $0}' | grep -P 'TTTAGTACACA' | cut -f 1 -d ' ' | sort | uniq > readlists/${SAMPLE}_ltr_integration_seq_read_ids.txt
done

Details of reference sequences and alignments produced

GRCh38 reference copied from: /storage1/fs1/bga/Active/gmsroot/gc2560/core/GRC-human-build38_human_95_38_U2AF1_fix/all_sequences.fa HTLV-1 reference obtained from: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/863/585/GCF_000863585.1_ViralProj15434/GCF_000863585.1_ViralProj15434_genomic.fna.gz

GRCh38 and HTLV-1 references catted together and BWA index and alignment done with BWA version 0.7.17-r1198-dirty (bryanfisk/bwa:latest)

isub -i 'bryanfisk/bwa:latest' -m 32 -n 8
source $WORKING_DIR/git/htlv_integration_sites/envs.txt
cd $WORKING_DIR

echo "${FASTQ_NAMES[@]}"

for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    echo -e "\nProcessing FASTQ: $FASTQ_NAME (R1 and R2)"
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')
    echo "Will name output using sample name: $SAMPLE"
    /usr/local/bwa/bwa mem -K 20000000 -t 8 -Y $WORKING_DIR/references/GRCh38+HTLV-1.fa $WORKING_DIR/fastqs/${FASTQ_NAME}R1_001.fastq.gz $WORKING_DIR/fastqs/${FASTQ_NAME}R2_001.fastq.gz | samtools view -o $WORKING_DIR/bams/${SAMPLE}.bam -Shb /dev/stdin
done

exit

Sorting and index BAMs

Alignments converted to bam, sorted, and indexed with samtools version 1.11

isub -i 'bryanfisk/bwa:latest' -m 32 -n 2
source $WORKING_DIR/git/htlv_integration_sites/envs.txt
cd $WORKING_DIR

for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')
    echo "Will name output using sample name: $SAMPLE"
    samtools sort -o $WORKING_DIR/bams/${SAMPLE}.sorted.bam -O BAM $WORKING_DIR/bams/${SAMPLE}.bam
    samtools index $WORKING_DIR/bams/${SAMPLE}.sorted.bam
done

exit

Duplicate marking step

Duplicates marked with picard version 2.22.8

cd $WORKING_DIR/tools
wget https://github.com/broadinstitute/picard/releases/download/2.22.8/picard.jar

isub -i 'bryanfisk/bwa:latest' -m 32 -n 2
source $WORKING_DIR/git/htlv_integration_sites/envs.txt
cd $WORKING_DIR

for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')
    echo "Will name output using sample name: $SAMPLE"
    java -Xmx16g -jar $WORKING_DIR/tools/picard.jar MarkDuplicates I=$WORKING_DIR/bams/${SAMPLE}.sorted.bam O=$WORKING_DIR/bams/${SAMPLE}.markedsorted.bam M=$WORKING_DIR/metrics_files/${SAMPLE}.markdup.metrics
    samtools index $WORKING_DIR/bams/${SAMPLE}.markedsorted.bam
done

exit

Flagstat step

Run samtools flagstats on dup marked BAMs

isub -m 32
source $WORKING_DIR/git/htlv_integration_sites/envs.txt
cd $WORKING_DIR

for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')
    echo "Will name output using sample name: $SAMPLE"
    samtools flagstat $WORKING_DIR/bams/${SAMPLE}.markedsorted.bam > $WORKING_DIR/metrics_files/${SAMPLE}.flagstat.metrics
done

exit

LTR integration site read filtering of BAM

Produce a version of the duplicate marked BAM that is limited to only those alignments involving reads that contained the characterstic integration site sequence (TTTAGTACACA|TGTGTACTAAA) identified above

isub -m 32
source $WORKING_DIR/git/htlv_integration_sites/envs.txt

cd $WORKING_DIR
rm -f tmp/*

for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')

    echo -e "\nProducing integration site read list filtered BAM for $SAMPLE"
    echo "samtools view -H bams/${SAMPLE}.markedsorted.bam > tmp/${SAMPLE}.markedsorted.bam.header"
    samtools view -H bams/${SAMPLE}.markedsorted.bam > tmp/${SAMPLE}.markedsorted.bam.header

    echo "samtools view bams/${SAMPLE}.markedsorted.bam | grep -F -f readlists/${SAMPLE}_ltr_integration_seq_read_ids.txt > tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.sam"
    samtools view bams/${SAMPLE}.markedsorted.bam | grep -F -f readlists/${SAMPLE}_ltr_integration_seq_read_ids.txt > tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.sam

    echo "cat tmp/${SAMPLE}.markedsorted.bam.header tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.sam | samtools view -Sb - > tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam"
    cat tmp/${SAMPLE}.markedsorted.bam.header tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.sam | samtools view -Sb - > tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam

    echo "samtools sort tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam -o bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam"
    samtools sort tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam -o bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam

    echo "samtools index bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam"
    samtools index bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam
done

identify reads involving a primary or supplementary alignment to the virus sequence (`NC_001436.1`)

isub -m 32
source $WORKING_DIR/git/htlv_integration_sites/envs.txt

cd $WORKING_DIR

for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')

    echo -e "\nProcessing sample: $SAMPLE"
    echo "Obtaining reads with supplemetary alignments to the virus"
    echo "samtools view -f 2048 bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam 'NC_001436.1' | cut -f 1 | sort | uniq > readlists/${SAMPLE}_supplementary_virus_hit_read_ids.txt"
    samtools view -f 2048 bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam 'NC_001436.1' | cut -f 1 | sort | uniq > readlists/${SAMPLE}_supplementary_virus_hit_read_ids.txt
    wc -l readlists/${SAMPLE}_supplementary_virus_hit_read_ids.txt

    echo -e "\nObtaining reads with primary alignments to the virus"
    echo "samtools view -F 256 bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam 'NC_001436.1' | cut -f 1 | sort | uniq > readlists/${SAMPLE}_primary_virus_hit_read_ids.txt"
    samtools view -F 256 bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam 'NC_001436.1' | cut -f 1 | sort | uniq > readlists/${SAMPLE}_primary_virus_hit_read_ids.txt
    wc -l readlists/${SAMPLE}_primary_virus_hit_read_ids.txt

    echo -e "\nCreating a unique list of reads with either supplementary or primary alignments to the virus"
    echo "cat readlists/${SAMPLE}_supplementary_virus_hit_read_ids.txt readlists/${SAMPLE}_primary_virus_hit_read_ids.txt | sort | uniq > readlists/${SAMPLE}_virus_hit_read_ids.txt"
    cat readlists/${SAMPLE}_supplementary_virus_hit_read_ids.txt readlists/${SAMPLE}_primary_virus_hit_read_ids.txt | sort | uniq > readlists/${SAMPLE}_virus_hit_read_ids.txt
    wc -l readlists/${SAMPLE}_virus_hit_read_ids.txt
    echo -e "\n"
done
exit

Use the viral alignment read list to produce filtered BAM files with only those reads that have such alignments

isub -m 32
source $WORKING_DIR/git/htlv_integration_sites/envs.txt

cd $WORKING_DIR

for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')
    echo -e "\nProducing viral read list filtered BAM for $SAMPLE"
    echo "samtools view -H bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam > tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam.header"
    samtools view -H bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam > tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam.header

    echo "samtools view bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam | grep -F -f readlists/${SAMPLE}_virus_hit_read_ids.txt > tmp/${SAMPLE}.markedsorted.viralreads.sam"
    samtools view bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam | grep -F -f readlists/${SAMPLE}_virus_hit_read_ids.txt > tmp/${SAMPLE}.markedsorted.viralreads.sam

    echo "cat tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam.header tmp/${SAMPLE}.markedsorted.viralreads.sam | samtools view -Sb - > bams/${SAMPLE}.markedsorted_with_hits_to_viral.bam"
    cat tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam.header tmp/${SAMPLE}.markedsorted.viralreads.sam | samtools view -Sb - > bams/${SAMPLE}.markedsorted_with_hits_to_viral.bam
done

exit

NOTE: It is possible that the above approach of limited to reads with a viral alignment could be too strict. A read may correspond to an integration event, have the characteristic LTR sequence, but not produce an alignment to the virus genome We should try the analysis, with and without this requirement and gauge impact

Use bedtools (v2.25.0) to create bed representations of the BAM alignments to facilitate integration site counting. At the same time apply filters to: require alignment, remove duplicates, prevent counting on the virus seq itself

Do this two ways: (1) with the marked-duplicate BAM, (2) with the BAM created from marked-duplicate BAM that also limits to viral hit reads produced above

(1) with the marked-duplicate BAM

isub -m 32
source $WORKING_DIR/git/htlv_integration_sites/envs.txt

cd $WORKING_DIR

rm -f tmp/*
for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')
    echo -e "\nProducing integration site counts $SAMPLE"
    echo "samtools view -H bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam > tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam.header"
    samtools view -H bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam > tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam.header
    echo "samtools view -f 1 -F 1024 -q 20 bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam > tmp/${SAMPLE}.markedsorted_filtered.sam"
    samtools view -f 1 -F 1024 -q 20 bams/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam > tmp/${SAMPLE}.markedsorted_filtered.sam
    echo "cat tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam.header tmp/${SAMPLE}.markedsorted_filtered.sam | samtools view -Sb - > tmp/${SAMPLE}.markedsorted_filtered.bam"
    cat tmp/${SAMPLE}.markedsorted_ltr_integration_seq_reads.bam.header tmp/${SAMPLE}.markedsorted_filtered.sam | samtools view -Sb - > tmp/${SAMPLE}.markedsorted_filtered.bam
    echo "samtools sort tmp/${SAMPLE}.markedsorted_filtered.bam -o bams/${SAMPLE}.markedsorted_filtered.bam"
    samtools sort tmp/${SAMPLE}.markedsorted_filtered.bam -o bams/${SAMPLE}.markedsorted_filtered.bam
    echo "samtools index bams/${SAMPLE}.markedsorted_filtered.bam"
    samtools index bams/${SAMPLE}.markedsorted_filtered.bam
    echo "bedtools bamtobed -i bams/${SAMPLE}.markedsorted_filtered.bam > beds/${SAMPLE}.markedsorted_filtered.bed"
    bedtools bamtobed -i bams/${SAMPLE}.markedsorted_filtered.bam > beds/${SAMPLE}.markedsorted_filtered.bed
    echo "bedtools merge -i beds/${SAMPLE}.markedsorted_filtered.bed -c 1 -o count | grep -v 'NC_001436.1' > beds/${SAMPLE}.markedsorted_filtered_merged.bed"
    bedtools merge -i beds/${SAMPLE}.markedsorted_filtered.bed -c 1 -o count | grep -v 'NC_001436.1' > beds/${SAMPLE}.markedsorted_filtered_merged.bed
    echo "cp beds/${SAMPLE}.markedsorted_filtered_merged.bed counts/${SAMPLE}.markedsorted_filtered_merged.bed.tsv"
    cp beds/${SAMPLE}.markedsorted_filtered_merged.bed counts/${SAMPLE}.markedsorted_filtered_merged.bed.tsv
done

exit

(2) with the BAM created from marked-duplicate BAM that also limits to viral hit reads produced above

isub -m 32
source $WORKING_DIR/git/htlv_integration_sites/envs.txt

cd $WORKING_DIR

rm -f tmp/*

for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')
    echo -e "\nProducing integration site counts $SAMPLE"
    echo "samtools view -H bams/${SAMPLE}.markedsorted_with_hits_to_viral.bam > tmp/${SAMPLE}.markedsorted_with_hits_to_viral.bam.header"
    samtools view -H bams/${SAMPLE}.markedsorted_with_hits_to_viral.bam > tmp/${SAMPLE}.markedsorted_with_hits_to_viral.bam.header
    echo "samtools view -f 1 -F 1024 -q 20 bams/${SAMPLE}.markedsorted_with_hits_to_viral.bam > tmp/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.sam"
    samtools view -f 1 -F 1024 -q 20 bams/${SAMPLE}.markedsorted_with_hits_to_viral.bam > tmp/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.sam
    echo "cat tmp/${SAMPLE}.markedsorted_with_hits_to_viral.bam.header tmp/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.sam | samtools view -Sb - > tmp/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bam"
    cat tmp/${SAMPLE}.markedsorted_with_hits_to_viral.bam.header tmp/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.sam | samtools view -Sb - > tmp/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bam
    echo "samtools sort tmp/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bam -o bams/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bam"
    samtools sort tmp/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bam -o bams/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bam
    echo "samtools index bams/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bam"
    samtools index bams/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bam
    echo "bedtools bamtobed -i bams/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bam > beds/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bed"
    bedtools bamtobed -i bams/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bam > beds/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bed
    echo "bedtools merge -i beds/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bed -c 1 -o count | grep -v 'NC_001436.1' > beds/${SAMPLE}.markedsorted_with_hits_to_viral_filtered_merged.bed"
    bedtools merge -i beds/${SAMPLE}.markedsorted_with_hits_to_viral_filtered.bed -c 1 -o count | grep -v 'NC_001436.1' > beds/${SAMPLE}.markedsorted_with_hits_to_viral_filtered_merged.bed
    echo "cp beds/${SAMPLE}.markedsorted_with_hits_to_viral_filtered_merged.bed counts/${SAMPLE}.markedsorted_with_hits_to_viral_filtered_merged.bed.tsv"
    cp beds/${SAMPLE}.markedsorted_with_hits_to_viral_filtered_merged.bed counts/${SAMPLE}.markedsorted_with_hits_to_viral_filtered_merged.bed.tsv
done

exit

Create single file with all counts for all samples to facilitate creation of visualizations

isub -m 32
source $WORKING_DIR/git/htlv_integration_sites/envs.txt

cd $WORKING_DIR

rm -f tmp/*

for FASTQ_NAME in "${FASTQ_NAMES[@]}"; do
    SAMPLE=$(echo $FASTQ_NAME | awk -F_ '{print $2}')
    awk -v sample="$SAMPLE" '{print $0 "\t" sample}' counts/${SAMPLE}.markedsorted_with_hits_to_viral_filtered_merged.bed.tsv > tmp/${SAMPLE}.markedsorted_with_hits_to_viral_filtered_merged.bed.tsv
done
echo -e "chromosome\tstart_pos\tend_pos\tcount\tsample" > tmp/header.tsv
cat tmp/header.tsv tmp/*markedsorted_with_hits_to_viral_filtered_merged.bed.tsv > counts/ALL.markedsorted_with_hits_to_viral_filtered_merged.bed.tsv

exit

Name		Name	Last commit message	Last commit date
Latest commit History 21 Commits
data		data
scripts		scripts
.gitignore		.gitignore
LICENSE		LICENSE
README.md		README.md
envs.txt		envs.txt

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Example of an HTLV-1 integration site analysis

High level questions

Samples in each batch

Set ENVs

Download the data

Get the file base names:

Investigate the four supplies possible integration characteristic sequences:

Create unique read lists of these read identities and store them for later use

Details of reference sequences and alignments produced

Sorting and index BAMs

Duplicate marking step

Flagstat step

LTR integration site read filtering of BAM

identify reads involving a primary or supplementary alignment to the virus sequence (`NC_001436.1`)

Use the viral alignment read list to produce filtered BAM files with only those reads that have such alignments

(1) with the marked-duplicate BAM

(2) with the BAM created from marked-duplicate BAM that also limits to viral hit reads produced above

Create single file with all counts for all samples to facilitate creation of visualizations

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Example of an HTLV-1 integration site analysis

High level questions

Samples in each batch

Set ENVs

Download the data

Get the file base names:

Investigate the four supplies possible integration characteristic sequences:

Create unique read lists of these read identities and store them for later use

Details of reference sequences and alignments produced

Sorting and index BAMs

Duplicate marking step

Flagstat step

LTR integration site read filtering of BAM

identify reads involving a primary or supplementary alignment to the virus sequence (NC_001436.1)

Use the viral alignment read list to produce filtered BAM files with only those reads that have such alignments

(1) with the marked-duplicate BAM

(2) with the BAM created from marked-duplicate BAM that also limits to viral hit reads produced above

Create single file with all counts for all samples to facilitate creation of visualizations

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identify reads involving a primary or supplementary alignment to the virus sequence (`NC_001436.1`)

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