Paraphase: HiFi-based caller for highly homologous genes

Many medically relevant genes fall into 'dark' regions where variant calling is limited due to high sequence homology with paralogs or pseudogenes. Paraphase is a Python tool that takes HiFi aligned BAMs as input (whole-genome or enrichment), phases haplotypes for genes of the same family, determines copy numbers and makes phased variant calls.

Paraphase takes all reads from a gene family, realigns to just the gene of interest and then phases them into haplotypes. This solves the problem of alignment difficulty due to sequence homology and allows us to examine all copies of genes in a gene family and call copy number changes and other variants.

Paraphase supports 161 segmental duplication regions in GRCh38. Among these, there are 11 medically relevant regions that are also supported in GRCh37/hg19:

• SMN1/SMN2 (spinal muscular atrophy)
• RCCX module
  • CYP21A2 (21-Hydroxylase-Deficient Congenital Adrenal Hyperplasia)
  • TNXB (Ehlers-Danlos syndrome)
  • C4A/C4B (relevant in autoimmune diseases)
• PMS2 (Lynch Syndrome)
• STRC (hereditary hearing loss and deafness)
• IKBKG (Incontinentia Pigmenti)
• NCF1 (chronic granulomatous disease; Williams syndrome)
• NEB (Nemaline myopathy)
• F8 (intron 22 inversion, Hemophilia A)
• CFC1 (heterotaxy syndrome)
• OPN1LW/OPN1MW (color vision deficiencies)
• HBA1/HBA2 (Alpha-Thalassemia)

Please check out our paper on its application to the gene SMN1 for more details about Paraphase.
Chen X, Harting J, Farrow E, et al. Comprehensive SMN1 and SMN2 profiling for spinal muscular atrophy analysis using long-read PacBio HiFi sequencing. The American Journal of Human Genetics. 2023;0(0). doi:10.1016/j.ajhg.2023.01.001

For whole-genome sequencing (WGS) data, we recommend >20X, ideally 30X, genome coverage. Low coverage or short read length could result in less accurate phasing, especially when gene copies are highly similar to each other. For hybrid capture-based enrichment data, a higher read depth (>50X) is recommended as the read length is generally shorter than WGS.

Contact

If you have suggestions or need assistance, please don't hesitate to reach out by email or open a GitHub issue.

Xiao Chen: [email protected]

Dependencies

• samtools
• minimap2

Installation

Paraphase can be installed through pip or conda:

pip install paraphase
# or
conda install -c conda-forge -c bioconda paraphase

Alternatively, Paraphase can be installed from GitHub.

git clone https://github.com/PacificBiosciences/paraphase
cd paraphase
python setup.py install

Running the program

paraphase -b input.bam -o output_directory -r genome_fasta

Alternatively when you have a list of bam files

paraphase -l list.txt -o output_directory -r genome_fasta

Required parameters:

• -b: Input BAM file or -l: text file listing BAM files one per line (a BAI file needs to exist in the same directory)
• -o: Output directory
• -r: Path to the reference genome fasta file

Please note that the input BAM should be one that's aligned to the ENTIRE reference genome (either GRCh38 or GRCh37/hg19), and this reference should NOT include ALT contigs. The fasta file of this reference genome should be provided to Paraphase with -r.

Optional parameters:

• -g: Region(s) to analyze, separated by comma. All supported regions will be analyzed if not specified. Please use region name, i.e. first column in the doc.
• -t: Number of threads.
• --genome: Genome reference build. Default is 38. If 37 or 19 is specified, Paraphase will run the analysis for GRCh37 or hg19, respectively (note that only 11 medically relevant regions are supported now for GRCh37/hg19).
• gene1only: If specified, variants calls will be made against the main gene only for SMN1, PMS2, STRC, NCF1 and IKBKG, see below.
• --novcf: If specified, no VCF files will be produced.
• --samtools: path to samtools. If the paths to samtools or minimap2 are not already in the PATH environment variable, they can be provided through the --samtools and --minimap2 parameters.
• --minimap2: path to minimap2

Interpreting the output

Paraphase produces a few output files in the directory specified by -o, with the sample ID as the prefix.

1. .vcf in sampleID_vcfs folder. A VCF file is written for each haplotype per gene family. There is also a _variants.vcf file containing merged variants from all haplotypes for each gene family. Note that this is not a diploid vcf as there are usually more than two copies of genes in a gene family in a sample.

As genes of the same family can be highly similar to each other in sequence and not easy to differentiate (at the sequence level or even at the functional level), variant calls are made against one selected "main" gene from the gene family (e.g. the functional gene is selected when the family has a gene and a pseudogene). In this way, all copies of the gene family can be evaluated for pathogenic variants and one can calculate the copy number of the functional genes in the family and hence infer the disease/carrier status.

Exceptions are SMN1 (paralog SMN2), PMS2 (pseudogene PMS2CL), STRC (pseudogene STRCP1), NCF1 (pseudogenes NCF1B and NCF1C) and IKBKG (pseudogene IKBKGP1), where gene differentiation is possible. In these families, haplotypes are assigned to each gene in the family, i.e. gene or paralog/pseudogene, and variants are called against the gene (or paralog/pseudogene) for the gene (or paralog/pseudogene) haplotypes, respectively. Variants calls can be made against the main gene only for these five families if --gene1only is specified.

2. _realigned_tagged.bam: This BAM file can be loaded into IGV for visualization of haplotypes (group reads by HP tag and color alignments by YC tag). All haplotypes are aligned against the main gene of interest. Tutorials/Examples are provided for medically relevant genes (See below).

3. .json: Output file summarizing haplotypes and variant calls for each gene family in each sample. In brief, a few generally used fields are explained below.

• final_haplotypes: phased haplotypes for all gene copies in a gene family
• total_cn: total copy number of the family (sum of gene and paralog/pseudogene)
• two_copy_haplotypes: haplotypes that are present in two copies based on depth. This happens when (in a small number of cases) two haplotypes are identical and we infer that there exist two of them instead of one by checking the read depth.
• haplotype_details: lists information about each haplotype
  • boundary: the boundary of the region that is resolved on the haplotype. This is useful when a haplotype is only partially phased.
• alleles_final: haplotypes phased into alleles. This is possible when the segmental duplication is in tandem.
• region_depth: median depth of the gene family (include all copies of gene and paralog/pseudogene)

Tutorials/Examples are provided for interpreting the json output and visualizing haplotypes for medically relevant genes listed below:

• SMN1/SMN2
• RCCX module (CYP21A2)
• PMS2
• STRC
• OPN1LW/OPN1MW
• HBA1/HBA2
• IKBKG
• F8
• NEB
• NCF1