Polymutt: a tool for calling polymorphism and de novo mutations in families for sequencing data

Introduction

• The program polymutt implemented a likelihood-based framework for calling single nucleotide variants and detecting de novo point mutation events in families for next-generation sequencing data. The program takes as input genotype likelihood format (GLF) files which can be generated following the Creation of GLF files instruction and outputs the result in the [VCF] format. The variant calling and de novo mutation detection are modelled jointly within families and can handle both nuclear and extended pedigrees without consanguinity loops. The input is a set of GLF files for each of family members and the relationships are specified through the .ped file.

• The evidence of variants and de novo mutations are assessed probabilistically. For a variant, the QUAL value is calculated as -10*log10(1-posterior(Variant | Data)) and for de novo mutation events a de novo quality (DQ) value is defined as log10(lk_denovo / lk_no_denovo) where lk_denovo and lk_no_denovo are the likelihoods of data allowing and disallowing de novo mutations respectively. Similarly, for each genotype, a genotype quality (GQ) value is defined as -10*log10(1-posterior(Genotype | Data)).

• Since unrelated individuals are kind of special case of families, unrelated individuals or a mixture of related and unrelated individuals can be handled.

• If some individuals in a family are not sequenced, this can be handled by setting the corresponding GLF file indices to zero for those family members who are not sequenced.

• See below for more details.

Usage

A command without any input will display the basic usage

polymutt

The following parameters are in effect:
                      pedfile :        test.ped (-pname)
                      datfile :        test.dat (-dname)
                 glfIndexFile :        test.gif (-gname)
             posterior cutoff :           0.900 (-c99.999)

Additional Options
      Map Quality Filter : --minMapQuality
            Depth Filter : --minDepth [150], --maxDepth [200],
                           --minPercSampleWithData [0.00]
    Scaled mutation rate : --theta [1.0e-03]
    Prior of ts/tv ratio : --tstv [2.00]
        de novo mutation : --denovo, --denovo_rate [0.00],
                           --denovo_tstv [0.50], --denovo_min_LLR [1.00]
  Optimization precision : --prec [1.0e-04]
      Multiple threading : --nthreads [4]
                  Output : --vcf [test.vcf], --gl_off

An example command for variant calling looks like the following:

polymutt -p in.ped -d in.dat -g glfIndexFile --vcf out.vcf --nthreads 4

An example command for de novo mutation detection is as follows:

polymutt -p in.ped -d in.dat -g glfIndexFile --denovo --rate_denovo 1.5e-08 --min_denovo_LLR 1.0 --vcf out.denovo.vcf --nthreads 4

Input files

Required input files are -p input.ped -d input.dat -g glfIndex files

• An example in.ped file looks like the following:

fam1 p1  0  0   1  1
fam1 p2  0  0   2  2
fam1 p3  p1 p2  1  3
fam2 p4  0  0   1  4
fam2 p5  0  0   2  5
fam2 p6  p4 p5  1  6
...

• An example in.dat file is like the following (for the 6th column above and in addition other traits/markers can be specified but will be ignored):

T GLF_Index

• An example glfIndex file is like the following and the numbers (except zeros) in the 6th column in the above in.ped file have to be present in the first column.

1  /home/me/sample1.glf
2  /home/me/sample2.glf
3  /home/me/sample3.glf
4  /home/me/sample4.glf
...

• If some of the members are not sequenced but are in the pedigree because of the relatedness with other members, the GLF_Index column (6th column) in the ped file should be set to zero
• For unrelated individuals, you can either (1) create a family for each unrelated individual as a founder or (2) put all unrelated individuals as founders in a single family.

Other options

Some of command line options are explained below and others are self-explanatory.

-c : minimum cutoff of posterior probability to output a variant [ Default: 0.5 ]
--theta : scaled mutation rate per site [Default: 0.001]
--tstv: prior of ts:tv ratio [Default: 2.0]
--nthreads : number of threads to run and it is recommended to use 4 threads for small number of input files [Default: 1']

--de_novo : a boolean flag to turn on de novo mutation detection. The following options take effect only when this flag is ON
--rate_denovo : mutation rate per haplotype per generation. [Default: 1e-08]
--tstv_denovo : the prior ts/tv ratio of de novo mutations. [Default: 2.0]
--min_denovo_LLR : minimum value of log10 likelihood ratio of allowing vs. disallowing de novo mutations in the data to output [Default: 1.0]

Output files

• The output file is a VCF file and the specification can be found [here]
• Since there is no standard to represent de novo mutations in the current VCF specification, actual genotypes (e.g. [ACGT]/[ACGT]) are output in the VCF file for de novo mutations.
• A summary about variant calling statistics is output to STDOUT and it may be redirected to a file for a record.

Summary of reference -- 9
Total Entry Count: 141213431 
Total Base Cout: 120124735
Total '0' Base Count:       137
Non-Polymorphic Count:   655457
Transition Count:      6556
Transversion Count:      3127
Other Polymorphism Count:         0
Filter counts:
       minMapQual 4550
       minTotalDepth 1089
       maxTotalDepth 736
Hard to call:         0
Skipped bases: 134

Creation of GLF files

• The current version performs variant calling and de novo mutation detection from files in the genotype likelihood format (GLF). In future versions we plan to take [SAM/BAM] files as input. See the following for instructions on how to create GLF files.
  • Download a modified version of samtools ( [samtools-hybrid] )
  • Prepare the reference genome in fasta format and sequence alignments in [SAM/BAM] format
  • Generate BAQ adjusted GLF files using the following command

samtools view -bh chr1.bam 1:0 | samtools calmd -Abr - human.v37.fa 2> /dev/null | samtools pileup - -g -f human.v37.fa > chr1.bam.glf

• For other functionalities please refer to the [samtools] website.

Download

The pre-compiled 64-bit binary executable for linux with test files can be downloaded here . Source code will be available to download soon.

Contact

For questions please contact the authors (Bingshan Li: bingshan@umich.edu or Goncalo Abecasis: goncalo@umich.edu)