## clone github repository and install hapfusion
git clone https://github.com/sjin09/hapfusion
cd hapfusion
/bin/bash install.sh
## use miniamp2 and samtools to align, sort (and merge) PacBio CCS read alignments
minimap2 "@RG\tSM:sample" -ax map-hifi --cs ref.fa pacbio.ccs.fastq.gz | samtools sort -o aln.sorted.bam # SM tag must be provided to retrieve sample ID
samtools merge *.sorted.bam | samtools sort -o aln.mergeSorted.bam - ## if there are multiple BAM files, merge and sort the BAM files
samtools view -bh -F 0x900 aln.sorted.bam > aln.primary_alignments.sorted.bam # select primary alignments
samtools index aln.sorted.bam
samtools index aln.primary_alignments.sorted.bam
## use deepvariant to call germline mutations
deepvariant.simg /opt/deepvariant/bin/run_deepvariant --model_type=PACBIO --ref ref.fa --reads=aln.primary_alignments.sorted.bam --output_vcf=germline.vcf
## use hapfusion to phase germline mutations
hapfusion phase --bam aln.primary_alignments.sorted.bam --vcf germline.vcf.bgz -o germline.phased.vcf
bgzip -c germline.phased.vcf > germline.phased.vcf.bgz
tabix -p vcf germline.phased.vcf.bgz
## call crossovers and gene conversions
hapfusion call -i aln.primary_alignments.sorted.bam --vcf germline.phased.vcf.bgz -o recombinations.txt
## plot crossovers and gene conversions
hapfusion plot -i aln.primary_alignments.sorted.bam --vcf germline.phased.vcf.bgz --fusion recombinations.txt -o pdfhapfusion leverages CCS base accuracy and read length to crossovers (CO), gene conversions (NCO) and complex gene conversions (CNCO).
Currently, hapfusion only accepts minimap2 CCS read alignment SAM/BAM file with a --cs tag, like the cigar string provides information about the matches and mismatches between the read and the reference genome. pbmm2 and ngmlr does not provide the --cs tag and as a result, pbmm2 and ngmlr SAM/BAM files are incompatible with himut. The --cs tag is favored over the cigar string because it offers a more elegant representation of the differences between the read and the reference genome (source: https://lh3.github.io/2018/03/27/the-history-the-cigar-x-operator-and-the-md-tag).
hapfusion accepts as input a BAM file with primary CCS read alignments and a VCF file with phased germline mutations and returns a file with crossovers and gene conversions.
## phase germline mutations
hapfusion phase --bam aln.primary_alignments.sorted.bam --vcf germline.vcf.bgz -o germline.phased.vcf
## call crossovers and gene conversions
hapfusion call -i aln.primary_alignments.sorted.bam --vcf germline.phased.vcf.bgz -o recombinations.txt
## plot crossovers and gene conversions
hapfusion plot -i aln.primary_alignments.sorted.bam --vcf germline.phased.vcf.bgz --fusion recombinations.txt -o pdfThe number of crossovers and gene conversions detcted from the sample is dependent on the number of phased hetSNPs, length of haplotype blocks and CCS read length.