Difference between revisions of "Polymutt: a tool for calling polymorphism and de novo mutations in families for sequencing data"

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== Introduction ==
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#REDIRECT [[Polymutt]]
* 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 | Creation of GLF files]] instruction and outputs the result in the [[http://www.1000genomes.org/node/101 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, --rate_denovo [0.00],
 
                            --tstv_denovo [2.00], --minLLR_denovo [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']
 
 
 
--denovo : 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: 1.5e-08'']
 
--tstv_denovo : the prior ts/tv ratio of ''de novo'' mutations. [''Default: 2.0'']
 
--minLLR_denovo : minimum value of log10 likelihood ratio of allowing vs. disallowing ''de novo'' mutations in the data to output [''Default: 1.0'']
 
 
--gl_off: not to output genotype likelihood values for each individual. Default is to output 3 GLs for polymorphisms and 10 GLs for de novo mutations
 
 
 
== Output files ==
 
* The output file is a VCF file and the specification can be found [[http://www.1000genomes.org/node/101 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 [[http://samtools.sourceforge.net/ SAM/BAM]] files as input. See the following for instructions on how to create GLF files.
 
** Download a modified version of samtools ( [[https://github.com/statgen/samtools-0.1.7a-hybrid samtools-hybrid]] )
 
** Prepare the reference genome in fasta format and sequence alignments in [[http://samtools.sourceforge.net/ 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  [[http://samtools.sourceforge.net/ samtools]] website.
 
 
 
== Download ==
 
The pre-compiled 64-bit binary executable for linux with test files can be [[Media:polymutt.tar.gz | downloaded]] here . Source code will be available to download soon.
 
 
 
== Contact ==
 
For questions please contact the authors (Bingshan Li:  [mailto:bingshan@umich.edu bingshan@umich.edu] or Goncalo Abecasis: [mailto:goncalo@umich.edu goncalo@umich.edu])
 

Latest revision as of 13:45, 3 December 2014

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