paraphase 2.2.0

paraphase: HiFi-based caller for highly homologous genes

Paraphase: HiFi-based caller for highly homologous genes

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

Paraphase supports the following genes:

• 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)

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 haplotypes are highly similar to each other in Exons 1-6. For hybrid capture-based enrichment data, a higher read depth (>50X) is recommended as the read length is generally shorter than WGS.

Currently Paraphase only works on GRCh38. Support for GRCh37 will be adde in the future.

Contact

There is a need for building consensus on how to report variants in segmental duplication regions, which could be complicated due to the frequent presence of copy number changes. If you have suggestions or need assistance, please don't hesitate to reach out by email or open a GitHub issue.

Xiao Chen: [e-mail]

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
• -o: Output directory
• -r: Path to the reference genome fasta file

Optional parameters:

• -g: Gene(s) to analyze. All supported genes will be analyzed if not specified.
• -t: Number of threads, used when -l is specified.
• -d: File listing average genome depth per sample, with two columns, sample ID and depth values, separated by tab or space. This saves run time by skipping the step to calculate genome depth.
• --novcf: no vcf output if specified.
• --samtools: path to samtools
• --minimap2: path to minimap2

The paths to samtools and minimap2 can be provided through the --samtools and --minimap2 parameters.

Interpreting the output

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

• _realigned_tagged.bam: This BAM file can be loaded into IGV for visualization of haplotypes, see haplotype visualization.
• .vcf: A VCF file is written for each haplotype, and there is also a _variants.vcf file containing merged variants from all haplotypes.
• .json: Main output file, summerizes haplotypes and variant calls for each sample. Details of the fields are explained below for each gene.

SMN1

• smn1_cn: copy number of SMN1, a null call indicates that Paraphase finds only one haplotype but depth does not unambiguously support a copy number of one or two.
• smn2_cn: copy number of SMN2, a null call indicates that Paraphase finds only one haplotype but depth does not unambiguously support a copy number of one or two.
• smn2_del78_cn: copy number of SMN2Δ7–8 (SMN2 with a deletion of Exon7-8)
• smn1_read_number: number of reads containing c.840C
• smn2_read_number: number of reads containing c.840T
• smn2_del78_read_number: number of reads containing the known deletion of Exon7-8 on SMN2
• smn1_haplotypes: phased SMN1 haplotypes
• smn2_haplotypes: phased SMN2 haplotypes
• smn2_del78_haplotypes: phased SMN2Δ7–8 haplotypes
• 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
  • variants: The variants contained in the haplotype, excluding those in homopolymer regions.
  • boundary: The boundary of the region that is resolved on the haplotype. This is useful when a haplotype is only partially phased.
  • haplogroup: The haplogroup that the haplotype is assigned to

RCCX, PMS2, NCF1, CFC1, STRC, IKBKG, NEB & F8

• total_cn: total copy number of the family (sum of gene and paralog/pseudogene)
• gene_cn: copy number of the gene of interest, when the gene and pseudogene can be easily distinguished with known sequence differences, as in PMS2/NCF1/STRC/IKBKG
• final_haplotypes: phased haplotypes
• 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.

Multiple copies of the repeat are phased inito alleles with read-based phasing in the case of RCCX/IKBKG/NEB. Additional output entries include:

• alleles_final: haplotypes phased into alleles

RCCX

More info fields on phasing haplotypes into alleles and annotation of CYP21A2:

• annotated_alleles: allele annotation for the CYP21A2 gene. This is only based on common gene-pseudogene (CYP21A2-CYP21A1P) conversions (P31L, IVS2-13A/C>G, G111Vfs, I173N, I237N, V238E, M240K, V282L, Q319X and R357W). Please refer to the vcfs for most thorough variant calling and annotation.
• ending_hap: the last copy of RCCX on each allele. Only these copies contain parts of TNXB (while the other copies contain TNXA)

IKBKG

• deletion_haplotypes: haplotypes carrying the 11.8kb deletion

F8

Additional output is included to report SVs that occur between the repeat regions:

• sv_called: reports deletions/duplications between int22h-1 and int22h-2, or inversions between int22h-1 and int22h-3