# Tagged with #phasing 2 documentation articles | 0 announcements | 5 forum discussions

## 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.

There are two types of GATK tools that are able to use pedigree (family structure) information:

### Tools that require a pedigree to operate

PhaseByTransmission and CalculateGenotypePosterior will not run without a properly formatted pedigree file. These tools are part of the Genotype Refinement workflow, which is documented here.

### Tools that are able to generate standard variant annotations

The two variant callers (HaplotypeCaller and the deprecated UnifiedGenotyper) as well as VariantAnnotator and GenotypeGVCFs are all able to use pedigree information if you request an annotation that involves population structure (e.g. Inbreeding Coefficient). To be clear though, the pedigree information is not used during the variant calling process; it is only used during the annotation step at the end.

If you already have VCF files that were called without pedigree information, and you want to add pedigree-related annotations (e.g to use Variant Quality Score Recalibration (VQSR) with the InbreedingCoefficient as a feature annotation), don't panic. Just run the latest version of the VariantAnnotator to re-annotate your variants, requesting any missing annotations, and make sure you pass your PED file to the VariantAnnotator as well. If you forget to provide the pedigree file, the tool will run successfully but pedigree-related annotations may not be generated (this behavior is different in some older versions).

The PED files used as input for these tools are based on PLINK pedigree files. The general description can be found here.

For these tools, the PED files must contain only the first 6 columns from the PLINK format PED file, and no alleles, like a FAM file in PLINK.

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The 2013 "best practices" workshop slides recommend running PhaseByTransmission followed by ReadBackedPhasing --respectPhaseInput.

1. The --respectPhaseInput option is not currently listed in the documentation. Does that mean that RBP now always respects phasing in the input VCF?

2. Does (or did) --respectPhaseInput cause phased sites in the input to be assumed correct, or are they just ignored? That is, does RBP --respectPhaseInput use the partial haplotypes from the input file as evidence?

Thanks! Douglas

I'm a bit confused regarding the new GATK version and new HC-functions. I'm trying to call haplotypes in a family of plants. I call Haplotypes using haplotype caller, then I want to run Read-backed phasing on the raw vcfs and then CalculateGenotypePosterios to add pedigree information. The CalculateGenotypePosterios-Walker seems to need the format Fields AC and AN, but they are not produced by the HaplotypeCaller. They used to be in earlier HC-Versions though...(?). How can I fix this? And is this a proper workflow at all? Is Read-backed phasing needed or has it become redundant with the new HC-Version being able to do physical phasing. Would it be "enough" to run HC for phasing and CalculateGenotypePosterios to add pedigree information? Anyhow the problem of missing ac and an fields remains. I would be greatful for some help on this.

Thsi is how a raw vcf produced by HC looks like

# CHROM POS ID REF ALT QUAL FILTER INFO FORMAT GfGa4742

GSVIVT01012145001 1 . G . . END=113 GT:DP:GQ:MIN_DP:PL 0/0:0:0:0:0,0,0 GSVIVT01012145001 114 . C . . END=164 GT:DP:GQ:MIN_DP:PL 0/0:172:99:164:0,120,1800 GSVIVT01012145001 165 . T C, 7732.77 . DP=175;MLEAC=2,0;MLEAF=1.00,0.00;MQ=60.00;MQ0=0 GT:AD:DP:GQ:PGT:PID:PL:SB 1/1:0,173,0:173:99:0|1:165_T_C:7761,521,0,7761,521,7761:0,0,165,8 GSVIVT01012145001 166 . G . . END=166 GT:DP:GQ:MIN_DP:PL 0/0:174:72:174:0,72,1080 GSVIVT01012145001 167 . T . . END=175 GT:DP:GQ:MIN_DP:PL 0/0:174:66:174:0,60,900 GSVIVT01012145001 176 . T . . END=191 GT:DP:GQ:MIN_DP:PL 0/0:174:57:173:0,57,855 GSVIVT01012145001 192 . A . . END=194 GT:DP:GQ:MIN_DP:PL 0/0:173:54:173:0,54,810 GSVIVT01012145001 195 . T . . END=199 GT:DP:GQ:MIN_DP:PL 0/0:174:51:173:0,51,765

And this is the Error Message I get

##### ERROR MESSAGE: Key AC found in VariantContext field INFO at GSVIVT01012145001:1 but this key isn't defined in the VCFHeader. We require all VCFs to have complete VCF headers by default.

Hi,

Is there a way that HaplotypeCaller (or another tool) can output phased genotypes in the VCF file?

Thanks! Andrew

Hello! I was wondering if the HaplotypeScore annotation was restored for HaplotypeCaller in GATK 2.6. Does it have to be called? (It's not included in my vcf file.) Moreover, all of the GT field designations have "/" instead of "|" which according to the following would mean that the results are still unphased:

"GT genotype, encoded as alleles values separated by either of ”/” or “|”, e.g. The allele values are 0 for the reference allele (what is in the reference sequence), 1 for the first allele listed in ALT, 2 for the second allele list in ALT and so on. For diploid calls examples could be 0/1 or 1|0 etc. For haploid calls, e.g. on Y, male X, mitochondrion, only one allele value should be given. All samples must have GT call information; if a call cannot be made for a sample at a given locus, ”.” must be specified for each missing allele in the GT field (for example ./. for a diploid). The meanings of the separators are: / : genotype unphased | : genotype phased" http://www.1000genomes.org/wiki/Analysis/Variant%20Call%20Format/vcf-variant-call-format-version-40

Also, is there a more detailed explanation than what's on the HaplotypeScore documentation page? How is the score determined in UnifiedGenotyper? Does the score have anything to do with phasing? Also, how is phasing achieved if only the 10bps surrounding the SNP are examined, regions which likely do not include other SNPs?

Thank you!

Hi,

I used Beagle to phase my data but for some indels, I have some probleme :

example :

Input vcf :

2       68599872        .       ATG     A       14.40   PASS    AC=1;AC1=1;AF=0.028


Input for beagle created by ProduceBeagleInput:

2:68599872 TG - 1.0000 0.0000 0.0000 ......


Output vcf created by BeagleOutputToVCF:

2       68599872        .       ATG     .       14.40   BGL_RM_WAS_-    AC1=1;AF1=0.02965.....


error message by CombineVariants:

MESSAGE: Badly formed variant context at location 68599872 in contig 2. Reference length must be at most one base shorter than location size


Can you help me?

Tipahine