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Kiran N' Bishwa edited this page Aug 14, 2018 · 5 revisions

Welcome to the phase-Extender wiki!

This tutorial provides the requisite knowledge for preparing required input files and running phase-Extender. phase-Extender is run with three different example sets and test files for the runs are provided in the appropriate links.

Part-A: Run phase extender.

Step 01: Prepare input files.

A) Convert VCF to haplotype file using VCF-Simplify:

python3 VCF-Simplify.py SimplifyVCF -toType haplotype -PI PI -PG PG -vcf RBphased_file.vcf -out haploype_file.txt

Note:

  • If RBphase information is represented by FORMAT fields other than "PI" and "PG", then it can be replaced accordingly.
  • "--unphased yes" flag can be raised to include the unphased genotypes. This parameter will not affect the phase extension, and is only included to keep the whole data intact.
  • run command python3 VCF-Simplify.py SimplifyVCF -h for more details on VCF and haplotype reference panel to table conversion.

B) Convert haplotype reference panel (VCF) to haplotype file:

python3 VCF-Simplify.py SimplifyVCF -toType haplotype --PI CHROM --PG GT --vcf RefPanel.vcf --out RefPanel_haploype.txt

Step 02: Run phase-Extender.

Parameters that are not called are set at default value.

  • Call for help -
python3 phase-Extender.py --help

  • Example test case 01 (with minimal parameters) -
    Use data from example 01 
python3 phase-Extender.py --input input_haplotype_file.txt --SOI ms02g --lods 10
  
# to write the computed LOD between two blocks to the output file
python3 phase-Extender.py --input input_haplotype_file.txt --SOI ms02g --writeLOD yes

#Output is stored in directory `ms02g_extended\`

  • Example test case 02 (multiple cases) -
    Use data from example 02 
# use 2 processes
# use 25 heterozygote SNPs (from each block) for ...
  # ... preparing transition matrix between two consecutive blocks
# set lods2 cutoff threshold at 10
# prepare descriptive statistics of the haplotype (initial vs. final) 
python3 phase-Extender.py --nt 2 --input haplotype_file_test02.txt --SOI ms02g --numHets 25 --culLH maxPd --hapStats yes --lods 10

# Output is stored in directory `ms02g_extended\`.


# to assign output to a different directory
python3 phase-Extender.py --input haplotype_file_test02.txt --SOI ms02g --output my_test
# Output is stored in directory `my_test\`.

# add "Reference Haplotype Panel" to the run
python3 phase-Extender.py --nt 2 --input haplotype_file_test02.txt --SOI ms02g --numHets 25 --culLH maxPd --hapStats yes --refHap refPanel_lyrata_test02.txt

# add "bed file" to the run
python3 phase-Extender.py --nt 1 --input haplotype_file_test02.txt --SOI ms02g --numHets 25 --culLH maxPd --hapStats yes --refHap refPanel_lyrata_test02.txt --bed bed_boundries.bed

# only use samples from "reference panel" to run phase extension
python3 phase-Extender.py --input haplotype_file_test02.txt --SOI ms02g --refHap refPanel_lyrata_test02.txt --useSample refHap

# only use specific samples to to run phase extension
# ** it is possible to mix sample names between reference panel and input haplotype file
python3 phase-Extender.py --input haplotype_file_test02.txt --SOI ms02g --useSample ms01e,ms02g,MA605,Sp76

  • Example test case 03 (using large dataset) -
    The data is ReadBackPhased haplotype (chr2 and chr3) from several samples of A. lyrata (Mayodan, Spiterstulen and F1 hybrids) used in my study. Click here for the dataset
    • input file is "inputHaplotype_chr2n3.txt".
    • output are in directory "ms02g_chr2n3_output"
python3 phase-Extender.py --nt 2 --input inputHaplotype_chr2n3.txt --SOI ms02g --output ms02g_chr2n3_output --numHets 25 --culLH 'maxPd' --hapStats yes --lods 10 --writeLOD yes --addMissingSites yes


Part-B: Interpreting phase-Extender's output

1. Interpreting phase extension between blocks (from output of test case 01) -

Initial phase state of sample ms02g in input file:

ms02g:PI    ms02g:PG_al
6     C|T
6     T|C
6     A|C
6     G|T
4     T|C
4     T|C
4     T|C
4     T|A

Extended phase state of sample ms02g (with computed LOD) in output file:

CHROM	POS	REF	all-alleles	ms02g:PI	ms02g:PG_al	log2odds
2	15881764	.	.	6	C|T	.
2	15881767	.	.	6	T|C	.
2	15881989	.	.	6	A|C	.
2	15882091	.	.	6	G|T	.
2	15882451	.	.	6	C|T	-73.30333
2	15882454	.	.	6	C|T	-73.30333
2	15882493	.	.	6	C|T	-73.30333
2       15882505        .       .       6       A|T     -73.30333

What does this tell us?

  • Our cutoff threshold was 10 and the computed lods between the two blocks is -73.3033.
  • Since, the |computed lods| > lods threshold phase-Extender proceeds with phase extension between blocks with PI (6 and 4).
  • Since, the compute lods is a negative value, the phase is exteded in alternate configuration.
  • So, if |computed lods| < lods threshold the phase state won't extend between two consecutive blocks.


2. Interpreting histogram plots (from output of test case 03) -\

So, when phase-Extender runs it will join two consecutive haplotype and will increase the size of the haplotype by number of variants within each haplotype, and also by length of the haplotype. Inversly, the total number of haplotype will decrease. This change can be compared by observing changes in the shape of the histogram (size by number of the haplotype) before vs. after phase extension.

Histogram of the haplotype size distribution before phase extension (contig 2 & 3) beforephaseextension!



Histogram of the haplotype size distribution after phase extension (contig 2 & 3) afterphaseextension!



Recursive application of haplotype phase extension

  • phase-Extender maynot be able to prepare a full length phased haplotype in one run. This is not the limitation but rather a intended feature in this tool. The reason is to provide flexibility and allow the user to control phase extension. A controllable haplotype extension is largely required for phase extension in emerging research model owing to smaller genotype samples, absence of reference panel and higher heterozygosity in the genome.\
  • The main idea is to first run phase-Extender with higher log2Odds cut off for several samples. Then merge the output of each sample to run another round of phase extension with concessive (lower) log2Odds cut off. When applied recursively we reduce the number of haplotypes and increase the length of haplotypes in each run.
  • To achieve full genomewide haplotype phasing there should be enough haplotype blocks (from other samples) bridging the haplotype breakpoint in the sample of interest.
  • So, controllable haplotype extension is a novel feature intended in phase-Extender. A full length recursive phase extension is illustrated in this link [phase Extender on recursive mode](some website??) using the bash script. (coming soon !)
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