analysis_BASIS-1Mb_assembly.Rmd

---
title: "Analysis for final 1.1 Mb BASIS assembly"
author: "Pierre Murat, Askar Kleefeldt"
date: "`r format(Sys.time(), '%d %B, %Y')`"
output: 
  html_document:
    toc: true
    toc_float: true
    toc_collapsed: true
    toc_depth: 3
    number_sections: true
    theme: lumen

---

# Prepare and index genomes
Human and bacterial genomes are catenated to generate a chimeric genomes used for alignment.

Human genome: Ensembl hg38/GRCh38
E. Coli: MDS42
Helper vectors: pKW20 and pLF118

```{bash, eval = F}
srun -c 112 --pty bash
# Extract contigs names
grep "^>" ./Genomes/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa > ./BASIS/Sequences/contig_names/hg38.seqname.txt
grep "^>" ./BASIS/Sequences/MDS42_WT_old_seq_better_annotation.fa > ./BASIS/Sequences/contig_names/MDS42.seqname.txt
grep "^>" ./BASIS/Sequences/p20_helper-plasmid_BASIS.fa > ./BASIS/Sequences/contig_names/p20.seqname.txt
grep "^>" ./BASIS/Sequences/pLF118_helper_plasmid_BACs.fa > ./BASIS/Sequences/contig_names/pLF118.seqname.txt

# Manually modify headers of bacterial fa files / re-run
# Join genomes and indexes
cat ./Genomes/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa \
    ./BASIS/Sequences/MDS42_WT_old_seq_better_annotation.fa \
    ./BASIS/Sequences/p20_helper-plasmid_BASIS.fa \
    ./BASIS/Sequences/pLF118_helper_plasmid_BACs.fa \
    > ./BASIS/Genomes/hg38.MDS42.fa
grep "^>" ./BASIS/Genomes/hg38.MDS42.fa > ./BASIS/Sequences/contig_names/hg38.MDS42.seqname.txt

./Software/bwa/bwa index ./BASIS/Genomes/hg38.MDS42.fa
samtools faidx ./BASIS/Genomes/hg38.MDS42.fa
./Software/gatk-4.2.5.0/gatk CreateSequenceDictionary -R ./BASIS/Genomes/hg38.MDS42.fa
```

# Create a reference haplotype
## Read alignment
All input adapted human BAC library BACs are aligned to the chimeric reference genome.

```{bash, eval = F}
srun -c 112 --pty bash
# Extract contigs names
grep "^>" ./Genomes/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa > ./BASIS/Sequences/contig_names/hg38.seqname.txt
grep "^>" ./BASIS/Sequences/MDS42_WT_old_seq_better_annotation.fa > ./BASIS/Sequences/contig_names/MDS42.seqname.txt
grep "^>" ./BASIS/Sequences/p20_helper-plasmid_BASIS.fa > ./BASIS/Sequences/contig_names/p20.seqname.txt
grep "^>" ./BASIS/Sequences/pLF118_helper_plasmid_BACs.fa > ./BASIS/Sequences/contig_names/pLF118.seqname.txt

# Manually modify headers of bacterial fa files / re-run
# Join genomes and indexes
cat ./Genomes/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa \
    ./BASIS/Sequences/MDS42_WT_old_seq_better_annotation.fa \
    ./BASIS/Sequences/p20_helper-plasmid_BASIS.fa \
    ./BASIS/Sequences/pLF118_helper_plasmid_BACs.fa \
    > ./BASIS/Genomes/hg38.MDS42.fa
grep "^>" ./BASIS/Genomes/hg38.MDS42.fa > ./BASIS/Sequences/contig_names/hg38.MDS42.seqname.txt

./Software/bwa/bwa index ./BASIS/Genomes/hg38.MDS42.fa
samtools faidx ./BASIS/Genomes/hg38.MDS42.fa
./Software/gatk-4.2.5.0/gatk CreateSequenceDictionary -R ./BASIS/Genomes/hg38.MDS42.fa

# Join FASTQ files
cat ./BASIS_v03/Data/fastq_BACs/BAC266_pH/9A9_S9_R1_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC267_rK/10A10_S10_R1_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC269_pH/11A11_S11_R1_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC270_rK/12A12_S12_R1_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC271_pH/13A13_S13_R1_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC272_rK/14A14_S14_R1_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC273_pH/15A15_S15_R1_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC274_rK/2A2_S2_R1_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC275_pH/86A86_S86_R1_001.fastq.gz \
    > ./BASIS_v03/Data/fastq_BACs/BACs.r1.fq.gz
    
cat ./BASIS_v03/Data/fastq_BACs/BAC266_pH/9A9_S9_R2_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC267_rK/10A10_S10_R2_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC269_pH/11A11_S11_R2_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC270_rK/12A12_S12_R2_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC271_pH/13A13_S13_R2_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC272_rK/14A14_S14_R2_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC273_pH/15A15_S15_R2_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC274_rK/2A2_S2_R2_001.fastq.gz \
    ./BASIS_v03/Data/fastq_BACs/BAC275_pH/86A86_S86_R2_001.fastq.gz \
    > ./BASIS_v03/Data/fastq_BACs/BACs.r2.fq.gz

# Count number of reads
zcat ./BASIS_v03/Data/fastq_BACs/BACs.r1.fq.gz | echo $((`wc -l`/4)) # 31,142,010 reads
zcat ./BASIS_v03/Data/fastq_BACs/BACs.r2.fq.gz | echo $((`wc -l`/4)) # 31,142,010 reads
# All libraries are well balanced

# align concatenated BAC fastq files to chimeric genome
Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.fa BASIS_v03/Data/fastq_BACs/BACs.r1.fq.gz BASIS_v03/Data/fastq_BACs/BACs.r2.fq.gz > BASIS_v03/Data/SAM/BACs.hg38.MDS42.sam

# sort and generate BAM file
Software/samtools-1.16.1/samtools sort -@ 4 -m 2G BASIS_v03/Data/SAM/BACs.hg38.MDS42.sam -o BASIS_v03/Data/BAM/BACs.hg38.MDS42.bam

# index BAM file
Software/samtools-1.16.1/samtools index BASIS_v03/Data/BAM/BACs.hg38.MDS42.bam

# count number of reads
Software/samtools-1.16.1/samtools view -c BASIS_v03/Data/BAM/BACs.hg38.MDS42.bam
# 62,737,268

# remove reads with multiple alignments
Software/samtools-1.16.1/samtools view -h BASIS_v03/Data/BAM/BACs.hg38.MDS42.bam | grep -v -e 'XA:Z:' -e 'SA:Z:' | Software/samtools-1.16.1/samtools view -b > BASIS_v03/Data/BAM/BACs.hg38.MDS42.unique.bam

# select properly paired reads
Software/samtools-1.16.1/samtools view -q 10 -F 1284 -f 0x02 -b BASIS_v03/Data/BAM/BACs.hg38.MDS42.unique.bam > BASIS_v03/Data/BAM/BACs.hg38.MDS42.paired.bam

# count number of reads
Software/samtools-1.16.1/samtools view -c BASIS_v03/Data/BAM/BACs.hg38.MDS42.paired.bam
# 51,283,822 reads

# Dump bacterial chromosomes
# Create an index file
awk '/^[0-9]*\t/ {printf("%s\t0\t%s\n",$1,$2);}' Genomes/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa.fai > BASIS_v03/Data/BAM/hum.index.bed

# filter only human chromosomes
Software/samtools-1.16.1/samtools view -L BASIS_v03/Data/BAM/hum.index.bed -o BASIS_v03/Data/BAM/BACs.only.hum.bam BASIS_v03/Data/BAM/BACs.hg38.MDS42.paired.bam

# index BACs.only.human.bam
Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAM/BACs.only.hum.bam

# Compute coverage using deeptool bam coverage
Software/miniconda3/envs/deeptools/bin/bamCoverage --bam BASIS_v03/Data/BAM/BACs.only.hum.bam -o BASIS_v03/Data/BW/BACs.only.hum.50nt.bw -of bigwig --binSize 50 --normalizeUsing CPM
```

## Base quality score recalibration (BCSR)
In order to call variants confidently, we need to recalibrate the base quality scores using an established pipeline available from GATK. Candidate “germline” short variants are then filtered by Variant Quality Score Recalibration (VQSR).

```{bash, eval = F}
# Mark duplicates and sort BAM
Software/gatk-4.3.0.0/gatk MarkDuplicates -I BASIS_v03/Data/BAM/BACs.only.hum.bam -O BASIS_v03/Data/BAMdup/BACs.only.hum.dup.bam -M BASIS_v03/Data/BAMdup/BACs.dup.metrics.txt

Software/gatk-4.3.0.0/gatk SortSam -I BASIS_v03/Data/BAMdup/BACs.only.hum.dup.bam -O BASIS_v03/Data/BAMdup/BACs.only.hum.dup.sort.bam -SO coordinate

# Add read groups (needed for picard/BQSR)
Software/gatk-4.3.0.0/gatk AddOrReplaceReadGroups -I BASIS_v03/Data/BAMdup/BACs.only.hum.dup.sort.bam -O BASIS_v03/Data/BAMdup/BACs.only.hum.dup.sort.group.bam -LB BAC_library -PL ILLUMINA -PU unknown -SM Normal

# Base quality score recalibration (BQSR)
# Generate recalibration table for BQSR
Software/gatk-4.3.0.0/gatk BaseRecalibrator -I BASIS_v03/Data/BAMdup/BACs.only.hum.dup.sort.group.bam -R Genomes/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa --known-sites ./ChromTrans/Dataset/GATK_resource/BQSR.known.sites.hg38.recode.sort.vcf -O BASIS_v03/Data/BQSR/BACs.only.hum.recal.data.table

# Apply BQSR
Software/gatk-4.3.0.0/gatk ApplyBQSR -R Genomes/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa -I BASIS_v03/Data/BAMdup/BACs.only.hum.dup.sort.group.bam --bqsr-recal-file BASIS_v03/Data/BQSR/BACs.only.hum.recal.data.table -O BASIS_v03/Data/BQSR/BACs.only.hum.bqsr.bam

Software/samtools-1.16.1/samtools index BASIS_v03/Data/BQSR/BACs.only.hum.bqsr.bam

```

## Define haplotype
Identify germline mutations with HaplotypeCaller and filtered using Variant Quality Score Recalibration (VQSR). VQSR uses an adaptive error model trained on “true sites” provided as input, typically HapMap and Omni 2.5M SNP chip array sites. Input are available from the GATK Resource Bundle, https://console.cloud.google.com/storage/browser/genomics-public-data/resources/broad/hg38/v0/ but need to be reformated to be used with Ensembl genomes.

```{bash, eval = F}
# Call variants
Software/gatk-4.3.0.0/gatk HaplotypeCaller \
    -R Genomes/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa \
    -I BASIS_v03/Data/BQSR/BACs.only.hum.bqsr.bam \
    -O BASIS_v03/Data/VCF/HAPLO/BACs.haplotype.vcf

# Build a recalibration model with VariantRecalibrator
Software/gatk-4.3.0.0/gatk VariantRecalibrator -R ./Genomes/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa -V BASIS_v03/Data/VCF/HAPLO/BACs.haplotype.vcf -AS --resource:hapmap,known=false,training=true,truth=true,prior=15.0 ./ChromTrans/Dataset/GATK_resource/hapmap_3.3.hg38.sites.recode.sort.vcf --resource:omni,known=false,training=true,truth=true,prior=12.0 ./ChromTrans/Dataset/GATK_resource/1000G_omni2.5.hg38.recode.sort.vcf --resource:1000G,known=false,training=true,truth=false,prior=10.0 ./ChromTrans/Dataset/GATK_resource/1000G_phase1.snps.high_confidence.hg38.recode.sort.vcf --resource:dbsnp,known=true,training=false,truth=false,prior=2.0 ./ChromTrans/Dataset/GATK_resource/Homo_sapiens_assembly38.dbsnp138.recode.sort.vcf -an QD -an MQ -an MQRankSum -an ReadPosRankSum -an FS -an SOR -mode SNP --max-gaussians 4 -O BASIS_v03/Data/VCF/HAPLO/BACs.output.AS.recal --tranches-file BASIS_v03/Data/VCF/HAPLO/BACs.output.AS.tranches --rscript-file BASIS_v03/Data/VCF/HAPLO/BACs.output.plots.AS.R

# Apply recalibration
Software/gatk-4.3.0.0/gatk ApplyVQSR -R ./Genomes/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa -V BASIS_v03/Data/VCF/HAPLO/BACs.haplotype.vcf -O BASIS_v03/Data/VCF/HAPLO/BACs.haplotype.VQSR.vcf -AS --truth-sensitivity-filter-level 99.0 --tranches-file BASIS_v03/Data/VCF/HAPLO/BACs.output.AS.tranches --recal-file BASIS_v03/Data/VCF/HAPLO/BACs.output.AS.recal -mode SNP

```

## Generate an alternative reference sequence
The previously identified germline variants are used to generate an alternative reference genome (FastaAlternateReferenceMaker). We first select variants on the regions covered by the BACs. To do so we look at region of chr21 with sufficient coverage to support domains covered by BACs.

```{R, eval = F}
library(rtracklayer)
BAC.cov.gr <- import.bw("/Volumes./BASIS_v03/Data/BW/BACs.only.hum.50nt.bw")
BAC.cov.chr21.gr <- BAC.cov.gr[seqnames(BAC.cov.gr) == "21"]

# Identify domains with more than 10 CPM per 50 nt and add an extra 10 kb on both side
BAC.domain.start <- min(as.data.frame(BAC.cov.chr21.gr[BAC.cov.chr21.gr$score >= 10])$start) - 10000
BAC.domain.end <- max(as.data.frame(BAC.cov.chr21.gr[BAC.cov.chr21.gr$score >= 10])$end) + 10000 

```

Replace the reference bases at variation sites for the interval covered by the BAC library.
WARNING: FastaAlternateReferenceMaker alters the name of the chromosomes, so we need to swap the contigs name.


```{bash, eval = F}
# make dict file using Picard and CreateSequenceDictionary
Software/gatk-4.3.0.0/gatk CreateSequenceDictionary -R BASIS_v03/Genomes/hg38.MDS42.fa -O BASIS_v03/Genomes/hg38.MDS42.fa.dict

# need to rename the dict file such that it does not end on .fa.dict but just on .dict

# rerun alternate reference maker
Software/gatk-4.3.0.0/gatk FastaAlternateReferenceMaker -R BASIS_v03/Genomes/hg38.MDS42.fa -O BASIS_v03/Genomes/hg38.MDS42.alternate.BAC.fa -V BASIS_v03/Data/VCF/HAPLO/BACs.haplotype.VQSR.vcf

grep "^>" BASIS_v03/Genomes/hg38.MDS42.alternate.BAC.fa > BASIS_v03/Sequences/contig_names/hg38.MDS42.alternate.seqname.txt

# rename fasta headers using a lookup table with seqkit (https://bioinf.shenwei.me/seqkit/)
sed '/^>/ s/ .*//' BASIS_v03/Genomes/hg38.MDS42.alternate.BAC.fa > BASIS_v03/Genomes/temp.fa
grep "^>" BASIS_v03/Genomes/temp.fa > BASIS_v03/Sequences/contig_names/temp.seqname.txt

```

```{R, eval = F}
# rename fasta headers using a lookup table with seqkit (https://bioinf.shenwei.me/seqkit/)
old.name <- read.table("/Volumes./BASIS_v03/Sequences/contig_names/temp.seqname.txt", sep = ",")
new.name <- read.table("/Volumes./BASIS_v03/Sequences/contig_names/hg38.MDS42.seqname.txt", sep = ",")

corr.df <- cbind.data.frame(gsub(">", "", old.name$V1), gsub(">", "", new.name$V1))
write.table(corr.df, "/Volumes./BASIS_v03/Sequences/contig_names/corr.table",
            sep = "\t", quote = F, col.names = F, row.names = F)
```

```{bash, eval = F}
# Use seqkit to replace the correct names
Software/miniconda3/bin/seqkit replace -p '([0-9]+)' -r '{kv}$2' -k BASIS_v03/Sequences/contig_names/corr.table BASIS_v03/Genomes/temp.fa > BASIS_v03/Genomes/hg38.MDS42.alternate.fa

grep "^>" BASIS_v03/Genomes/hg38.MDS42.alternate.fa > BASIS_v03/Sequences/contig_names/hg38.MDS42.alternate.seqname.txt

# Index alternate genome
Software/bwa/bwa index BASIS_v03/Genomes/hg38.MDS42.alternate.fa

```

# Somatic variant calling
## Align BACs and BASIS samples to alternate chromosome

```{bash, eval = F}

# aligning pool of BACs
Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa BASIS_v03/Data/fastq_BACs/BACs.r1.fq.gz BASIS_v03/Data/fastq_BACs/BACs.r2.fq.gz > BASIS_v03/Data/SAM/BACs.hg38.MDS42.alternate.sam

# aligning individual BACs
Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa BASIS_v03/Data/fastq_BACs/BAC266_pH/9A9_S9_R1_001.fastq.gz BASIS_v03/Data/fastq_BACs/BAC266_pH/9A9_S9_R2_001.fastq.gz > BASIS_v03/Data/SAM/BAC266.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa BASIS_v03/Data/fastq_BACs/BAC267_rK/10A10_S10_R1_001.fastq.gz BASIS_v03/Data/fastq_BACs/BAC267_rK/10A10_S10_R2_001.fastq.gz > BASIS_v03/Data/SAM/BAC267.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa BASIS_v03/Data/fastq_BACs/BAC269_pH/11A11_S11_R1_001.fastq.gz BASIS_v03/Data/fastq_BACs/BAC269_pH/11A11_S11_R2_001.fastq.gz > BASIS_v03/Data/SAM/BAC269.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa BASIS_v03/Data/fastq_BACs/BAC270_rK/12A12_S12_R1_001.fastq.gz BASIS_v03/Data/fastq_BACs/BAC270_rK/12A12_S12_R2_001.fastq.gz > BASIS_v03/Data/SAM/BAC270.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa BASIS_v03/Data/fastq_BACs/BAC271_pH/13A13_S13_R1_001.fastq.gz BASIS_v03/Data/fastq_BACs/BAC271_pH/13A13_S13_R2_001.fastq.gz > BASIS_v03/Data/SAM/BAC271.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa BASIS_v03/Data/fastq_BACs/BAC272_rK/14A14_S14_R1_001.fastq.gz BASIS_v03/Data/fastq_BACs/BAC272_rK/14A14_S14_R2_001.fastq.gz > BASIS_v03/Data/SAM/BAC272.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa BASIS_v03/Data/fastq_BACs/BAC273_pH/15A15_S15_R1_001.fastq.gz BASIS_v03/Data/fastq_BACs/BAC273_pH/15A15_S15_R2_001.fastq.gz > BASIS_v03/Data/SAM/BAC273.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa BASIS_v03/Data/fastq_BACs/BAC274_rK/2A2_S2_R1_001.fastq.gz BASIS_v03/Data/fastq_BACs/BAC274_rK/2A2_S2_R2_001.fastq.gz > BASIS_v03/Data/SAM/BAC274.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa BASIS_v03/Data/fastq_BACs/BAC275_pH/86A86_S86_R1_001.fastq.gz BASIS_v03/Data/fastq_BACs/BAC275_pH/86A86_S86_R2_001.fastq.gz > BASIS_v03/Data/SAM/BAC275.hg38.MDS42.alternate.sam

# aligning BASIS samples
Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
BASIS_v03/Data/fastq_BASIS/BASIS_1_180/SLX-21919.i701_i516.HWJGYBGXL.s_1.r_1.fq.gz \
BASIS_v03/Data/fastq_BASIS/BASIS_1_180/SLX-21919.i701_i516.HWJGYBGXL.s_1.r_2.fq.gz \
> BASIS_v03/Data/SAM/BASIS_1_180.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
BASIS_v03/Data/fastq_BASIS/BASIS_2_340/79A79_S79_R1_001.fastq.gz \
BASIS_v03/Data/fastq_BASIS/BASIS_2_340/79A79_S79_R2_001.fastq.gz \
> BASIS_v03/Data/SAM/BASIS_2_340.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
BASIS_v03/Data/fastq_BASIS/BASIS_3_503/52A52_S52_R1_001.fastq.gz \
BASIS_v03/Data/fastq_BASIS/BASIS_3_503/52A52_S52_R2_001.fastq.gz \
> BASIS_v03/Data/SAM/BASIS_3_503.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
BASIS_v03/Data/fastq_BASIS/BASIS_4_570/329D41_S329_R1_001.fastq.gz \
BASIS_v03/Data/fastq_BASIS/BASIS_4_570/329D41_S329_R2_001.fastq.gz \
> BASIS_v03/Data/SAM/BASIS_4_570.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
BASIS_v03/Data/fastq_BASIS/BASIS_5_670/308D20_S308_R1_001.fastq.gz \
BASIS_v03/Data/fastq_BASIS/BASIS_5_670/308D20_S308_R2_001.fastq.gz \
> BASIS_v03/Data/SAM/BASIS_5_670.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
BASIS_v03/Data/fastq_BASIS/BASIS_6_790/300D12_S300_R1_001.fastq.gz \
BASIS_v03/Data/fastq_BASIS/BASIS_6_790/300D12_S300_R2_001.fastq.gz \
> BASIS_v03/Data/SAM/BASIS_6_790.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
BASIS_v03/Data/fastq_BASIS/BASIS_7_910/9A9_S9_R1_001.fastq.gz \
BASIS_v03/Data/fastq_BASIS/BASIS_7_910/9A9_S9_R2_001.fastq.gz \
> BASIS_v03/Data/SAM/BASIS_7_910.hg38.MDS42.alternate.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
BASIS_v03/Data/fastq_BASIS/BASIS_8_1060_23-8/17A17_S17_R1_001.fastq.gz \
BASIS_v03/Data/fastq_BASIS/BASIS_8_1060_23-8/17A17_S17_R2_001.fastq.gz \
> BASIS_v03/Data/SAM/BASIS_8_1060_23-8.hg38.MDS42.alternate.sam

```

## Process all aligned data
Data are then processed as previously using ./BASIS/Scripts/Read_processing_SLURM.sh script (PM). Shell script is reported below:

```{bash, eval = F}
#!/bin/bash -l
for SAM in ./BASIS_v03/Data/SAM/*.hg38.MDS42.alternate.sam
do
# Recover file names
FILE=${SAM%.hg38.MDS42.alternate.sam}
SAMPLE=${FILE##*/}
echo $SAMPLE
# Sort and generate BAM file
./Software/samtools-1.16.1/samtools sort -@ 4 -m 2G ./BASIS_v03/Data/SAM/$SAMPLE.hg38.MDS42.alternate.sam -o ./BASIS_v03/Data/BAM/$SAMPLE.hg38.MDS42.alternate.bam

# Index BAM file
./Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAM/$SAMPLE.hg38.MDS42.alternate.bam

# Generate stats
./Software/samtools-1.16.1/samtools idxstats ./BASIS_v03/Data/BAM/$SAMPLE.hg38.MDS42.alternate.bam > ./BASIS_v03/Data/IDXSTATS/$SAMPLE.hg38.MDS42.alternate.idxstats.txt

# Remove reads with multiple alignments
./Software/samtools-1.16.1/samtools view -h ./BASIS_v03/Data/BAM/$SAMPLE.hg38.MDS42.alternate.bam | grep -v -e 'XA:Z:' -e 'SA:Z:' | ./Software/samtools-1.16.1/samtools view -b > ./BASIS_v03/Data/BAM/$SAMPLE.hg38.MDS42.alternate.unique.bam

# Select properly paired reads
./Software/samtools-1.16.1/samtools view -q 10 -F 1284 -f 0x02 -b ./BASIS_v03/Data/BAM/$SAMPLE.hg38.MDS42.alternate.unique.bam > ./BASIS_v03/Data/BAM/$SAMPLE.hg38.MDS42.alternate.paired.bam

./Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAM/$SAMPLE.hg38.MDS42.alternate.paired.bam

# Recompute stats
./Software/samtools-1.16.1/samtools idxstats ./BASIS_v03/Data/BAM/$SAMPLE.hg38.MDS42.alternate.paired.bam > ./BASIS_v03/Data/IDXSTATS/$SAMPLE.hg38.MDS42.alternate.paired.idxstats.txt

# Dump bacterial chromosomes
./Software/samtools-1.16.1/samtools view -L ./BASIS_v03/Data/BAM/hum.index.bed \
                                                          -o ./BASIS_v03/Data/BAM/$SAMPLE.alternate.only.hum.bam \
                                                             ./BASIS_v03/Data/BAM/$SAMPLE.hg38.MDS42.alternate.paired.bam 

./Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAM/$SAMPLE.alternate.only.hum.bam

# Compute coverage using deeptool bam coverage
./Software/miniconda3/envs/deeptools/bin/bamCoverage \
      --bam ./BASIS_v03/Data/BAM/$SAMPLE.alternate.only.hum.bam \
      -o    ./BASIS_v03/Data/BW/$SAMPLE.alternate.only.hum.50nt.bw \
      -of bigwig \
      --binSize 50 \
      --normalizeUsing CPM

done
```

## Mark duplicates and group reads
Data are then processed as previously using the ./BASIS/Scripts/Picard_mark_SLURM.sh script. Shell script is reported below:

```{bash, eval = F}
#!/bin/bash -l
for BAM in ./BASIS_v03/Data/BAM/*.alternate.only.hum.bam
do
# Recover file names
FILE=${BAM%.alternate.only.hum.bam}
SAMPLE=${FILE##*/}
echo $SAMPLE
# Mark duplicates and sort BAM
./Software/gatk-4.3.0.0/gatk MarkDuplicates \
    -I ./BASIS_v03/Data/BAM/$SAMPLE.alternate.only.hum.bam \
    -O ./BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.bam \
    -M ./BASIS_v03/Data/BAMdup/$SAMPLE.dup.metrics.txt
./Software/gatk-4.3.0.0/gatk SortSam \
    -I ./BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.bam \
    -O ./BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.bam \
    -SO coordinate
# Add read groups (needed for Mutect2)
./Software/gatk-4.3.0.0/gatk AddOrReplaceReadGroups \
    -I ./BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.bam \
    -O ./BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.group.bam \
    -LB $SAMPLE \
    -PL ILLUMINA \
    -PU unknown \
    -SM $SAMPLE
done
```

## Candidate short variants calling
Candidate short variants are called with Mutect2 with the tumor (= BASIS samples) with matched normal (= BACs).

```{bash, eval = F}
# Create a sequence dictionary file for the alternative genome
Software/gatk-4.3.0.0/gatk CreateSequenceDictionary -R ./BASIS_v03/Genomes/hg38.MDS42.alternate.fa

# Check read group information
Software/samtools-1.16.1/samtools view -H ./BASIS_v03/Data/BAMdup/BACs.alternate.only.hum.dup.sort.group.bam | grep '^@RG'
Software/samtools-1.16.1/samtools view -H ./BASIS_v03/Data/BAMdup/BASIS_8_1060_22-D4_newNGS.alternate.only.hum.dup.sort.group.bam | grep '^@RG'

# Index BAM files (of all relevant samples)
Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAMdup/BACs.alternate.only.hum.dup.sort.group.bam 
Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAMdup/BASIS_1_180.alternate.only.hum.dup.sort.group.bam
Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAMdup/BASIS_2_340.alternate.only.hum.dup.sort.group.bam
Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAMdup/BASIS_3_503.alternate.only.hum.dup.sort.group.bam
Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAMdup/BASIS_4_570.alternate.only.hum.dup.sort.group.bam
Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAMdup/BASIS_5_670.alternate.only.hum.dup.sort.group.bam
Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAMdup/BASIS_6_790.alternate.only.hum.dup.sort.group.bam
Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAMdup/BASIS_7_910.alternate.only.hum.dup.sort.group.bam
Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAMdup/BASIS_8_1060_23-8.alternate.only.hum.dup.sort.group.bam

# Run Mutect2 (Tumor with matched normal mode)
# BASIS samples
Software/gatk-4.3.0.0/gatk Mutect2 \
  -R ./BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
  -I ./BASIS_v03/Data/BAMdup/BACs.alternate.only.hum.dup.sort.group.bam \
  -I ./BASIS_v03/Data/BAMdup/BASIS_1_180.alternate.only.hum.dup.sort.group.bam -normal BACs \
  -O ./BASIS_v03/Data/VCF/SOMA/BASIS_1_180.mutect2.vcf

Software/gatk-4.3.0.0/gatk Mutect2 \
  -R ./BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
  -I ./BASIS_v03/Data/BAMdup/BACs.alternate.only.hum.dup.sort.group.bam \
  -I ./BASIS_v03/Data/BAMdup/BASIS_2_340.alternate.only.hum.dup.sort.group.bam -normal BACs \
  -O ./BASIS_v03/Data/VCF/SOMA/BASIS_2_340.mutect2.vcf

Software/gatk-4.3.0.0/gatk Mutect2 \
  -R ./BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
  -I ./BASIS_v03/Data/BAMdup/BACs.alternate.only.hum.dup.sort.group.bam \
  -I ./BASIS_v03/Data/BAMdup/BASIS_3_503.alternate.only.hum.dup.sort.group.bam -normal BACs \
  -O ./BASIS_v03/Data/VCF/SOMA/BASIS_3_503.mutect2.vcf

Software/gatk-4.3.0.0/gatk Mutect2 \
  -R ./BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
  -I ./BASIS_v03/Data/BAMdup/BACs.alternate.only.hum.dup.sort.group.bam \
  -I ./BASIS_v03/Data/BAMdup/BASIS_4_570.alternate.only.hum.dup.sort.group.bam -normal BACs \
  -O ./BASIS_v03/Data/VCF/SOMA/BASIS_4_570.mutect2.vcf

Software/gatk-4.3.0.0/gatk Mutect2 \
  -R ./BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
  -I ./BASIS_v03/Data/BAMdup/BACs.alternate.only.hum.dup.sort.group.bam \
  -I ./BASIS_v03/Data/BAMdup/BASIS_5_670.alternate.only.hum.dup.sort.group.bam -normal BACs \
  -O ./BASIS_v03/Data/VCF/SOMA/BASIS_5_670.mutect2.vcf

Software/gatk-4.3.0.0/gatk Mutect2 \
  -R ./BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
  -I ./BASIS_v03/Data/BAMdup/BACs.alternate.only.hum.dup.sort.group.bam \
  -I ./BASIS_v03/Data/BAMdup/BASIS_6_790.alternate.only.hum.dup.sort.group.bam -normal BACs \
  -O ./BASIS_v03/Data/VCF/SOMA/BASIS_6_790.mutect2.vcf

Software/gatk-4.3.0.0/gatk Mutect2 \
  -R ./BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
  -I ./BASIS_v03/Data/BAMdup/BACs.alternate.only.hum.dup.sort.group.bam \
  -I ./BASIS_v03/Data/BAMdup/BASIS_7_910.alternate.only.hum.dup.sort.group.bam -normal BACs \
  -O ./BASIS_v03/Data/VCF/SOMA/BASIS_7_910.mutect2.vcf
  
Software/gatk-4.3.0.0/gatk Mutect2 \
  -R ./BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
  -I ./BASIS_v03/Data/BAMdup/BACs.alternate.only.hum.dup.sort.group.bam \
  -I ./BASIS_v03/Data/BAMdup/BASIS_8_1060_23-8.alternate.only.hum.dup.sort.group.bam -normal BACs \
  -O ./BASIS_v03/Data/VCF/SOMA/BASIS_8_1060_23-8.mutect2.vcf

```

## Filter short variants
Candidate short variants are then filtered using the shell script ./BASIS/Scripts/Mutect_filter_SLURM.sh and ./BASIS/Scripts/Mutect_filter_SLURM.2.sh, reported below:

```{bash, eval = F}
#!/bin/bash -l
for VCF in ./BASIS_v03/Data/VCF/SOMA/*.mutect2.vcf
do
# Recover file names
FILE=${VCF%.mutect2.vcf}
SAMPLE=${FILE##*/}
echo $SAMPLE
# Filter Mutect2 VCF
./Software/gatk-4.3.0.0/gatk FilterMutectCalls \
   -V ./BASIS_v03/Data/VCF/SOMA/$SAMPLE.mutect2.vcf \
   -R ./BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
   -O ./BASIS_v03/Data/VCF/SOMA/$SAMPLE.mutect2.filter.vcf
# Generate table from VCF
./Software/gatk-4.3.0.0/gatk VariantsToTable \
     -V ./BASIS_v03/Data/VCF/SOMA/$SAMPLE.mutect2.filter.vcf \
     -F CHROM -F POS -F REF -F ALT -F FILTER -GF AF -GF GT \
     -O ./BASIS_v03/Data/VCF/SOMA/$SAMPLE.mutect2.filter.tsv
done
```

## Mutation analysis / curation

```{R, eval = F}
#R
library(dplyr)
# Load all detected somatic mutations
BASIS.files <- list.files(path = "/Volumes./BASIS_v03/Data/VCF/SOMA", pattern = "\\.mutect2.filter.tsv")
BASIS.samples <- gsub(".mutect2.filter.tsv", "", BASIS.files)
BASIS.path <- paste("/Volumes./BASIS_v03/Data/VCF/SOMA", BASIS.files, sep = "/")
BASIS.vcf <- tibble()
for (i in 1:length(BASIS.path)) {
  print(BASIS.samples[i])
  vcf.i <- read.table(BASIS.path[i], header = T) %>% mutate(SAMPLE = BASIS.samples[i])
  colnames(vcf.i) <- c("CHROM", "POS", "REF", "ALT", "FILTER", "BAC.AF", "BAC.GT", "BASIS.AF", "BASIS.GT", "SAMPLE")
  BASIS.vcf <- rbind(BASIS.vcf, vcf.i)
}
BASIS.vcf <- BASIS.vcf %>% filter(CHROM == 21 & POS >= 32545200 & POS <= 33615150)
BASIS.ligth.vcf <- BASIS.vcf %>% filter(BASIS.AF >= 0.1) %>% dplyr::select(CHROM, POS, REF, ALT) %>% unique() %>% filter(REF != "TRUE")
BASIS.true.vcf <- BASIS.vcf %>% filter(POS %in% BASIS.ligth.vcf$POS) %>% arrange(POS)
# Save vcf summary
write.csv(BASIS.true.vcf, "/Volumes./BASIS_v03/Data/VCF/SOMA.summary.csv")
# The resulting file is manually modified to report additional information
```

## Haplotype calling against alternate reference for additional verification
The alternate reference genome does not implement all SNV/INDELs. Around 200 SNV/INDEL per Mb are retained, requiring manual screening. For additional verification, we perform haplotype calling (germline mutation calling) against alternate fasta reference of both BACs and BASIS samples of interest (1.1 Mb). This yields all variants of the BACs and of BASIS samples with respect to the alternate reference, allowing to compare these two lists against each other to ensure that none of the called variants is missed.

```{bash, eval = F}
#!/bin/bash -l
for BAM in ./BASIS_v03/Data/BAM/BACs_alternate_only_hum_bam/*.alternate.only.hum.bam
do
# Recover file names
FILE=${BAM%.alternate.only.hum.bam}
SAMPLE=${FILE##*/}
echo $SAMPLE

# Mark duplicates and sort BAM
Software/gatk-4.3.0.0/gatk MarkDuplicates \
    -I BASIS_v03/Data/BAM/BACs_alternate_only_hum_bam/$SAMPLE.alternate.only.hum.bam \
    -O BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.bam \
    -M BASIS_v03/Data/BAMdup/$SAMPLE.dup.metrics.txt

Software/gatk-4.3.0.0/gatk SortSam \
    -I BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.bam \
    -O BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.bam \
    -SO coordinate

# Add read groups (needed for picard/BQSR)
Software/gatk-4.3.0.0/gatk AddOrReplaceReadGroups \
    -I BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.bam \
    -O BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.group.bam \
    -LB BACs -PL ILLUMINA -PU unknown -SM Normal

Software/samtools-1.16.1/samtools index BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.group.bam

# Call variants (germline variants)
Software/gatk-4.3.0.0/gatk HaplotypeCaller \
    -R BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    -I BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.group.bam \
    -O BASIS_v03/Data/VCF/HAPLO_alternate/$SAMPLE.alternate.haplotype.vcf
done
```

Similarly run for BASIS samples.

```{bash, eval = F}
#!/bin/bash -l
for BAM in ./BASIS_v03/Data/BAM/BASIS_alternate_only_hum_bam/*.alternate.only.hum.bam
do
# Recover file names
FILE=${BAM%.alternate.only.hum.bam}
SAMPLE=${FILE##*/}
echo $SAMPLE

# Mark duplicates and sort BAM
Software/gatk-4.3.0.0/gatk MarkDuplicates \
    -I BASIS_v03/Data/BAM/BASIS_alternate_only_hum_bam/$SAMPLE.alternate.only.hum.bam \
    -O BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.bam \
    -M BASIS_v03/Data/BAMdup/$SAMPLE.dup.metrics.txt

Software/gatk-4.3.0.0/gatk SortSam \
    -I BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.bam \
    -O BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.bam \
    -SO coordinate

# Add read groups (needed for picard/BQSR)
Software/gatk-4.3.0.0/gatk AddOrReplaceReadGroups \
    -I BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.bam \
    -O BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.group.bam \
    -LB BACs -PL ILLUMINA -PU unknown -SM Normal

Software/samtools-1.16.1/samtools index BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.group.bam

# Call variants (germline variants)
Software/gatk-4.3.0.0/gatk HaplotypeCaller \
    -R BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    -I BASIS_v03/Data/BAMdup/$SAMPLE.alternate.only.hum.dup.sort.group.bam \
    -O BASIS_v03/Data/VCF/HAPLO_alternate/$SAMPLE.alternate.haplotype.vcf
    
# write VCF file to table
./Software/gatk-4.3.0.0/gatk VariantsToTable \
  -V BASIS_v03/Data/VCF/HAPLO_alternate/$SAMPLE.alternate.haplotype.vcf \
  -O BASIS_v03/Data/VCF/HAPLO_alternate/$SAMPLE.alternate.haplotype.tsv

done
```


```{bash, eval = F}
# write VCF file to table
./Software/gatk-4.3.0.0/gatk VariantsToTable \
  -V BASIS_v03/Data/VCF/HAPLO_alternate/BAC266.alternate.haplotype.vcf \
  -O BASIS_v03/Data/VCF/HAPLO_alternate/BAC266.alternate.haplotype.tsv
  
./Software/gatk-4.3.0.0/gatk VariantsToTable \
  -V BASIS_v03/Data/VCF/HAPLO_alternate/BAC267.alternate.haplotype.vcf \
  -O BASIS_v03/Data/VCF/HAPLO_alternate/BAC267.alternate.haplotype.tsv

./Software/gatk-4.3.0.0/gatk VariantsToTable \
  -V BASIS_v03/Data/VCF/HAPLO_alternate/BAC269.alternate.haplotype.vcf \
  -O BASIS_v03/Data/VCF/HAPLO_alternate/BAC269.alternate.haplotype.tsv

./Software/gatk-4.3.0.0/gatk VariantsToTable \
  -V BASIS_v03/Data/VCF/HAPLO_alternate/BAC270.alternate.haplotype.vcf \
  -O BASIS_v03/Data/VCF/HAPLO_alternate/BAC270.alternate.haplotype.tsv

./Software/gatk-4.3.0.0/gatk VariantsToTable \
  -V BASIS_v03/Data/VCF/HAPLO_alternate/BAC271.alternate.haplotype.vcf \
  -O BASIS_v03/Data/VCF/HAPLO_alternate/BAC271.alternate.haplotype.tsv

./Software/gatk-4.3.0.0/gatk VariantsToTable \
  -V BASIS_v03/Data/VCF/HAPLO_alternate/BAC272.alternate.haplotype.vcf \
  -O BASIS_v03/Data/VCF/HAPLO_alternate/BAC272.alternate.haplotype.tsv

./Software/gatk-4.3.0.0/gatk VariantsToTable \
  -V BASIS_v03/Data/VCF/HAPLO_alternate/BAC273.alternate.haplotype.vcf \
  -O BASIS_v03/Data/VCF/HAPLO_alternate/BAC273.alternate.haplotype.tsv

./Software/gatk-4.3.0.0/gatk VariantsToTable \
  -V BASIS_v03/Data/VCF/HAPLO_alternate/BAC274.alternate.haplotype.vcf \
  -O BASIS_v03/Data/VCF/HAPLO_alternate/BAC274.alternate.haplotype.tsv

./Software/gatk-4.3.0.0/gatk VariantsToTable \
  -V BASIS_v03/Data/VCF/HAPLO_alternate/BAC275.alternate.haplotype.vcf \
  -O BASIS_v03/Data/VCF/HAPLO_alternate/BAC275.alternate.haplotype.tsv

```

Analyse variants called with Haplotype caller; filter out all the variants that are also called in the BACs. All remaining variants need manual verification.

```{R, eval = F}
#R
library(dplyr)
# Load all detected germline mutations for BACs against the alternate reference
BAC.files <- list.files(path = "./BASIS_v03/Data/VCF/HAPLO_alternate",
                        pattern = "\\.alternate.haplotype.tsv")
BAC.samples <- gsub(".alternate.haplotype.tsv", "", BAC.files)
BAC.path <- paste("./BASIS_v03/Data/VCF/HAPLO_alternate", BAC.files, sep = "/")
BAC.vcf <- tibble()
for (i in 1:length(BAC.path)) {
  print(BAC.samples[i])
  vcf.i <- read.table(BAC.path[i], header = T) %>% mutate(SAMPLE = BAC.samples[i])
  BAC.vcf <- rbind(BAC.vcf, vcf.i)
}

BAC.filter.vcf <- BAC.vcf %>% filter(CHROM == 21 & POS >= 32545200 & POS <= 33615150)

# Load all detected germline mutations for BASIS samples against the alternate reference
BASIS.files <- list.files(path = "./BASIS_v03/Data/VCF/HAPLO_alternate/BASIS",
                        pattern = "\\.alternate.haplotype.tsv")
BASIS.samples <- gsub(".alternate.haplotype.tsv", "", BASIS.files)
BASIS.path <- paste("./BASIS_v03/Data/VCF/HAPLO_alternate/BASIS", BASIS.files, sep = "/")
BASIS.vcf <- tibble()
for (i in 1:length(BASIS.path)) {
  print(BASIS.samples[i])
  vcf.i <- read.table(BASIS.path[i], header = T) %>% mutate(SAMPLE = BASIS.samples[i])
  BASIS.vcf <- rbind(BASIS.vcf, vcf.i)
}

BASIS.filter.vcf <- BASIS.vcf %>% filter(CHROM == 21 & POS >= 32545200 & POS <= 33615150)

# create individual data frames for BASIS sample
BASIS.filter.B238.vcf <- BASIS.filter.vcf %>% filter(SAMPLE == "BASIS_8_1060_23-8")

# compare each individual BASIS sample against the full collection of variants called for BACs
# only retain unique called variants that are not seen in BACs
BASIS.unique.B238.vcf <- anti_join(BASIS.filter.B238.vcf, BAC.filter.vcf, by = "POS")

write.csv(BASIS.unique.B238.vcf, "/Volumes./BASIS_v03/Data/VCF/HAPLO_alternate/B238.HAPLO.summary.csv")
```

# Structural variant detection

## Long-read sequencing data
Alignment of long-read sequencing data of BACs and BASIS samples to alternative reference genome.

```{bash, eval = F}

# find names of folders and files
cd ./BASIS_v03/Data/fastq_BACs_nanopore/
find -maxdepth 2 -type f | sort -h > BACS_fastq_names.txt

cd ./

# align Nanopore sequencing data of all BACs to new alternate reference genome v03
./guppy/bin/minimap2 \
    -ax map-ont BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    BASIS_v03/Data/fastq_BACs_nanopore/01_BASIS_BAC_266_160_pH/BASIS_BAC_266_160_barcode11.fastq \
    > BASIS_v03/Data/SAM/nanopore/BAC266.hg38.MDS42.alternate.sam
    
./guppy/bin/minimap2 \
    -ax map-ont BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    BASIS_v03/Data/fastq_BACs_nanopore/02_BASIS_BAC_267_187_rK/BASIS_BAC_267_187_barcode12.fastq \
    > BASIS_v03/Data/SAM/nanopore/BAC267.hg38.MDS42.alternate.sam

./guppy/bin/minimap2 \
    -ax map-ont BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    BASIS_v03/Data/fastq_BACs_nanopore/03_BASIS_BAC_269_195_pH/BASIS_BAC_269_195_barcode13.fastq \
    > BASIS_v03/Data/SAM/nanopore/BAC269.hg38.MDS42.alternate.sam

./guppy/bin/minimap2 \
    -ax map-ont BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    BASIS_v03/Data/fastq_BACs_nanopore/04_BASIS_BAC_270_258_rK/BASIS_BAC_270_258_barcode14.fastq \
    > BASIS_v03/Data/SAM/nanopore/BAC270.hg38.MDS42.alternate.sam

./guppy/bin/minimap2 \
    -ax map-ont BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    BASIS_v03/Data/fastq_BACs_nanopore/05_BASIS_BAC_271_298_pH/BASIS_BAC_271_298_barcode15.fastq \
    > BASIS_v03/Data/SAM/nanopore/BAC271.hg38.MDS42.alternate.sam

./guppy/bin/minimap2 \
    -ax map-ont BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    BASIS_v03/Data/fastq_BACs_nanopore/06_BASIS_BAC_272_371_rK/BASIS_BAC_272_371_barcode16.fastq \
    > BASIS_v03/Data/SAM/nanopore/BAC272.hg38.MDS42.alternate.sam

./guppy/bin/minimap2 \
    -ax map-ont BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    BASIS_v03/Data/fastq_BACs_nanopore/07_BASIS_BAC_273_413_pH/BASIS_BAC_273_413_barcode17.fastq \
    > BASIS_v03/Data/SAM/nanopore/BAC273.hg38.MDS42.alternate.sam

./guppy/bin/minimap2 \
    -ax map-ont BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    BASIS_v03/Data/fastq_BACs_nanopore/08_BASIS_BAC_274_468_rK/BAC274_rK_sHBA468_barcode04.fastq \
    > BASIS_v03/Data/SAM/nanopore/BAC274.hg38.MDS42.alternate.sam

./guppy/bin/minimap2 \
    -ax map-ont BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    BASIS_v03/Data/fastq_BACs_nanopore/09_BASIS_BAC_275_260_pH/BAC275_pH_sHBA260_barcode05.fastq \
    > BASIS_v03/Data/SAM/nanopore/BAC275.hg38.MDS42.alternate.sam


# find names of folders and files
cd ./BASIS_v03/Data/fastq_BASIS_nanopore/
find -maxdepth 2 -type f | sort -h > BASIS_fastq_names.txt

cd ./

# align Nanopore sequencing data of BASIS sample to new alternate reference genome v03

./guppy/bin/minimap2 \
    -ax map-ont BASIS_v03/Genomes/hg38.MDS42.alternate.fa \
    BASIS_v03/Data/fastq_BASIS_nanopore/BASIS_1060_23-8/BASIS_23-8_barcode19.fastq \
    > BASIS_v03/Data/SAM/nanopore/BASIS_1060_23-8.hg38.MDS42.alternate.sam

```

```{bash, eval = F}
#!/bin/bash -l
for SAM in ./BASIS_v03/Data/SAM/nanopore/*.hg38.MDS42.alternate.sam
do
# Recover file names
FILE=${SAM%.hg38.MDS42.alternate.sam}
SAMPLE=${FILE##*/}
echo $SAMPLE

Software/samtools-1.16.1/samtools view \
    -bS ./BASIS_v03/Data/SAM/nanopore/$SAMPLE.hg38.MDS42.alternate.sam \
    > ./BASIS_v03/Data/BAM/nanopore/$SAMPLE.hg38.MDS42.alternate.bam
    
Software/samtools-1.16.1/samtools sort \
    ./BASIS_v03/Data/BAM/nanopore/$SAMPLE.hg38.MDS42.alternate.bam \
    > ./BASIS_v03/Data/BAM/nanopore/$SAMPLE.hg38.MDS42.alternate.sorted.bam

Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAM/nanopore/$SAMPLE.hg38.MDS42.alternate.sorted.bam

# Dump bacterial chromosomes
./Software/samtools-1.16.1/samtools view -L ./BASIS_v03/Data/BAM/hum.index.bed \
                                                          -o ./BASIS_v03/Data/BAM/nanopore/$SAMPLE.alternate.only.hum.bam \
                                                          ./BASIS_v03/Data/BAM/nanopore/$SAMPLE.hg38.MDS42.alternate.sorted.bam 

./Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAM/nanopore/$SAMPLE.alternate.only.hum.bam

# Compute coverage using deeptool bam coverage
./Software/miniconda3/envs/deeptools/bin/bamCoverage \
      --bam ./BASIS_v03/Data/BAM/nanopore/$SAMPLE.alternate.only.hum.bam \
      -o    ./BASIS_v03/Data/BW/nanopore/$SAMPLE.alternate.only.hum.50nt.bw \
      -of bigwig \
      --binSize 50 \
      --normalizeUsing CPM

done
```

## Final alignment for vis

For visualisation, only region of interest was extracted, and following for SV were implemented:
32725783	BAC266/267	    DEL:87
33157357	BAC271	        INS:380-390
33322295	BAC273	        DEL:2517
33475438	BAC275	        INS:9.5kb

This manually corrected reference was used to align all BASIS samples.

```{bash, eval = F}
# indexing genome
Software/bwa/bwa index BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa
Software/samtools-1.16.1/samtools faidx BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa

# align BASIS samples with bwa
Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa \
  BASIS_v03/Data/fastq_BASIS/BASIS_1_180/5A5_S5_R1_001.fastq.gz \
  BASIS_v03/Data/fastq_BASIS/BASIS_1_180/5A5_S5_R2_001.fastq.gz \
  > BASIS_v03/Data/SAM/chr21_SV_implemented/BASIS_1_180.hg38.MDS42.alternate.chr21.curated.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa \
  BASIS_v03/Data/fastq_BASIS/BASIS_2_340/2A2_S2_R1_001.fastq.gz \
  BASIS_v03/Data/fastq_BASIS/BASIS_2_340/2A2_S2_R2_001.fastq.gz \
  > BASIS_v03/Data/SAM/chr21_SV_implemented/BASIS_2_340.hg38.MDS42.alternate.chr21.curated.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa \
  BASIS_v03/Data/fastq_BASIS/BASIS_3_503/6A6_S6_R1_001.fastq.gz \
  BASIS_v03/Data/fastq_BASIS/BASIS_3_503/6A6_S6_R2_001.fastq.gz \
  > BASIS_v03/Data/SAM/chr21_SV_implemented/BASIS_3_503.hg38.MDS42.alternate.chr21.curated.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa \
  BASIS_v03/Data/fastq_BASIS/BASIS_4_570/3A3_S3_R1_001.fastq.gz \
  BASIS_v03/Data/fastq_BASIS/BASIS_4_570/3A3_S3_R2_001.fastq.gz \
  > BASIS_v03/Data/SAM/chr21_SV_implemented/BASIS_4_570.hg38.MDS42.alternate.chr21.curated.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa \
  BASIS_v03/Data/fastq_BASIS/BASIS_5_670/7A7_S7_R1_001.fastq.gz \
  BASIS_v03/Data/fastq_BASIS/BASIS_5_670/7A7_S7_R2_001.fastq.gz \
  > BASIS_v03/Data/SAM/chr21_SV_implemented/BASIS_5_670.hg38.MDS42.alternate.chr21.curated.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa \
  BASIS_v03/Data/fastq_BASIS/BASIS_6_790/4A4_S4_R1_001.fastq.gz \
  BASIS_v03/Data/fastq_BASIS/BASIS_6_790/4A4_S4_R2_001.fastq.gz \
  > BASIS_v03/Data/SAM/chr21_SV_implemented/BASIS_6_790.hg38.MDS42.alternate.chr21.curated.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa \
  BASIS_v03/Data/fastq_BASIS/BASIS_7_910/9A9_S9_R1_001.fastq.gz \
  BASIS_v03/Data/fastq_BASIS/BASIS_7_910/9A9_S9_R2_001.fastq.gz \
  > BASIS_v03/Data/SAM/chr21_SV_implemented/BASIS_7_910.hg38.MDS42.alternate.chr21.curated.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa \
  BASIS_v03/Data/fastq_BASIS/BASIS_7-5_940_17-3/1A1_S1_R1_001.fastq.gz \
  BASIS_v03/Data/fastq_BASIS/BASIS_7-5_940_17-3/1A1_S1_R2_001.fastq.gz \
  > BASIS_v03/Data/SAM/chr21_SV_implemented/BASIS_7-5_940_17-3.hg38.MDS42.alternate.chr21.curated.sam

Software/bwa/bwa mem -M -t 7 BASIS_v03/Genomes/hg38.MDS42.alternate.chr21_ROI_v03_SV_curated_implemented.fa \
  BASIS_v03/Data/fastq_BASIS/BASIS_8_1060_23-8/17A17_S17_R1_001.fastq.gz \
  BASIS_v03/Data/fastq_BASIS/BASIS_8_1060_23-8/17A17_S17_R2_001.fastq.gz \
  > BASIS_v03/Data/SAM/chr21_SV_implemented/BASIS_8_1060_23-8.hg38.MDS42.alternate.chr21.curated.sam

```


```{bash, eval = F}
#!/bin/bash -l
for SAM in ./BASIS_v03/Data/SAM/chr21_SV_implemented/*.hg38.MDS42.alternate.chr21.curated.sam
do
# Recover file names
FILE=${SAM%.hg38.MDS42.alternate.chr21.curated.sam}
SAMPLE=${FILE##*/}
echo $SAMPLE

# Sort and generate BAM file
./Software/samtools-1.16.1/samtools sort -@ 4 -m 2G ./BASIS_v03/Data/SAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.sam -o ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.bam

# Index BAM file
./Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.bam

# Generate stats
./Software/samtools-1.16.1/samtools idxstats ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.bam > ./BASIS_v03/Data/IDXSTATS/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.idxstats.txt

# Remove reads with multiple alignments
./Software/samtools-1.16.1/samtools view -h ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.bam | grep -v -e 'XA:Z:' -e 'SA:Z:' | ./Software/samtools-1.16.1/samtools view -b > ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.unique.bam

# Select properly paired reads
./Software/samtools-1.16.1/samtools view -q 10 -F 1284 -f 0x02 -b ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.unique.bam > ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.paired.bam

./Software/samtools-1.16.1/samtools index ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.paired.bam

# Recompute stats
./Software/samtools-1.16.1/samtools idxstats ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.paired.bam > ./BASIS_v03/Data/IDXSTATS/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.idxstats.txt

# Compute coverage using deeptool bam coverage
./Software/miniconda3/envs/deeptools/bin/bamCoverage \
      --bam ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.paired.bam \
      -o    ./BASIS_v03/Data/BW/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.paired.1nt.bw \
      -of bigwig \
      --binSize 1 \
      --normalizeUsing CPM

done
```

```{bash, eval = F}
#!/bin/bash -l
for SAM in ./BASIS_v03/Data/SAM/chr21_SV_implemented/*.hg38.MDS42.alternate.chr21.curated.sam
do
# Recover file names
FILE=${SAM%.hg38.MDS42.alternate.chr21.curated.sam}
SAMPLE=${FILE##*/}
echo $SAMPLE

# Compute coverage using deeptool bam coverage with 50 nt bin size
./Software/miniconda3/envs/deeptools/bin/bamCoverage \
      --bam ./BASIS_v03/Data/BAM/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.paired.bam \
      -o    ./BASIS_v03/Data/BW/chr21_SV_implemented/$SAMPLE.hg38.MDS42.alternate.chr21.curated.paired.50nt.bw \
      -of bigwig \
      --binSize 50 \
      --normalizeUsing CPM
done
```