Read-backed Phasing

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Read-backed Phasing

Example and Command Line Arguments

For a complete, detailed argument reference, refer to the GATK document page here

Introduction

The biological unit of inheritance from each parent in a diploid organism is a set of single chromosomes, so that a diploid organism contains a set of pairs of corresponding chromosomes. The full sequence of each inherited chromosome is also known as a haplotype. It is critical to ascertain which variants are associated with one another in a particular individual. For example, if an individual's DNA possesses two consecutive heterozygous sites in a protein-coding sequence, there are two alternative scenarios of how these variants interact and affect the phenotype of the individual. In one scenario, they are on two different chromosomes, so each one has its own separate effect. On the other hand, if they co-occur on the same chromosome, they are thus expressed in the same protein molecule; moreover, if they are within the same codon, they are highly likely to encode an amino acid that is non-synonymous (relative to the other chromosome). The ReadBackedPhasing program serves to discover these haplotypes based on high-throughput sequencing reads.

The first step in phasing is to call variants ("genotype calling") using a SAM/BAM file of reads aligned to the reference genome -- this results in a VCF file. Using the VCF file and the SAM/BAM reads file, the ReadBackedPhasing tool considers all reads within a Bayesian framework and attempts to find the local haplotype with the highest probability, based on the reads observed.

The local haplotype and its phasing is encoded in the VCF file as a "|" symbol (which indicates that the alleles of the genotype correspond to the same order as the alleles for the genotype at the preceding variant site). For example, the following VCF indicates that SAMP1 is heterozygous at chromosome 20 positions 332341 and 332503, and the reference base at the first position (A) is on the same chromosome of SAMP1 as the alternate base at the latter position on that chromosome (G), and vice versa (G with C):

#CHROM  POS ID  REF ALT QUAL    FILTER  INFO    FORMAT  SAMP1   
chr20   332341  rs6076509   A   G   470.60  PASS    AB=0.46;AC=1;AF=0.50;AN=2;DB;DP=52;Dels=0.00;HRun=1;HaplotypeScore=0.98;MQ=59.11;MQ0=0;OQ=627.69;QD=12.07;SB=-145.57    GT:DP:GL:GQ 0/1:46:-79.92,-13.87,-84.22:99
chr20   332503  rs6133033   C   G   726.23  PASS    AB=0.57;AC=1;AF=0.50;AN=2;DB;DP=61;Dels=0.00;HRun=1;HaplotypeScore=0.95;MQ=60.00;MQ0=0;OQ=894.70;QD=14.67;SB=-472.75    GT:DP:GL:GQ:PQ  1|0:60:-110.83,-18.08,-149.73:99:126.93

The per-sample per-genotype PQ field is used to provide a Phred-scaled phasing quality score based on the statistical Bayesian framework employed for phasing. Note that for cases of homozygous sites that lie in between phased heterozygous sites, these homozygous sites will be phased with the same quality as the next heterozygous site.

Limitations:

• ReadBackedPhasing doesn't currently support insertions, deletions, or multi-nucleotide polymorphisms.
• Input VCF files should only be for diploid organisms.

More detailed aspects of semantics of phasing in the VCF format

• The "|" symbol is used for each sample to indicate that each of the alleles of the genotype in question derive from the same haplotype as each of the alleles of the genotype of the same sample in the previous NON-FILTERED variant record. That is, rows without FILTER=PASS are essentially ignored in the read-backed phasing (RBP) algorithm.
• Note that the first heterozygous genotype record in a pair of haplotypes will necessarily have a "/" - otherwise, they would be the continuation of the preceding haplotypes.
• A homozygous genotype is always "appended" to the preceding haplotype. For example, any 0/0 or 1/1 record is always converted into 0|0 and 1|1.
• RBP attempts to phase a heterozygous genotype relative the preceding HETEROZYGOUS genotype for that sample. If there is sufficient read information to deduce the two haplotypes (for that sample), then the current genotype is declared phased ("/" changed to "|") and assigned a PQ that is proportional to the estimated Phred-scaled error rate. All homozygous genotypes for that sample that lie in between the two heterozygous genotypes are also assigned the same PQ value (and remain phased).
• If RBP cannot phase the heterozygous genotype, then the genotype remains with a "/", and no PQ score is assigned. This site essentially starts a new section of haplotype for this sample.

For example, consider the following records from the VCF file:

#CHROM  POS ID  REF ALT QUAL    FILTER  INFO    FORMAT  SAMP1   SAMP2
chr1    1   .   A   G   99  PASS    .   GT:GL:GQ    0/1:-100,0,-100:99  0/1:-100,0,-100:99
chr1    2   .   A   G   99  PASS    .   GT:GL:GQ:PQ 1|1:-100,0,-100:99:60   0|1:-100,0,-100:99:50
chr1    3   .   A   G   99  PASS    .   GT:GL:GQ:PQ 0|1:-100,0,-100:99:60   0|0:-100,0,-100:99:60
chr1    4   .   A   G   99  FAIL    .   GT:GL:GQ    0/1:-100,0,-100:99  0/1:-100,0,-100:99
chr1    5   .   A   G   99  PASS    .   GT:GL:GQ:PQ 0|1:-100,0,-100:99:70   1|0:-100,0,-100:99:60
chr1    6   .   A   G   99  PASS    .   GT:GL:GQ:PQ 0/1:-100,0,-100:99  1|1:-100,0,-100:99:70
chr1    7   .   A   G   99  PASS    .   GT:GL:GQ:PQ 0|1:-100,0,-100:99:80   0|1:-100,0,-100:99:70
chr1    8   .   A   G   99  PASS    .   GT:GL:GQ:PQ 0|1:-100,0,-100:99:90   0|1:-100,0,-100:99:80

The proper interpretation of these records is that SAMP1 has the following haplotypes at positions 1-5 of chromosome 1:

1. AGAAA
2. GGGAG

And two haplotypes at positions 6-8:

1. AAA
2. GGG

And, SAMP2 has the two haplotypes at positions 1-8:

1. AAAAGGAA
2. GGAAAGGG

• Note that we have excluded the non-PASS SNP call (at chr1:4), thus assuming that both samples are homozygous reference at that site.