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This page documents how to perform variant calling from low-coverage sequencing data using glfmultiples and thunder. The pipeline was originally developed by [mailto:yunli@med.unc.edu Yun Li] for the 1000 Genomes Low Coverage Pilot Project.
This page documents how to perform variant calling from low-coverage sequencing data using glfmultiples and thunder. The pipeline was originally developed by [mailto:yunli@med.unc.edu Yun Li] and for [mailto:goncalo@umich.edu Goncalo Abecasis] the 1000 Genomes Low Coverage Pilot Project.
== Input Data ==
== Input Data ==
To get started, you will need glf files in the standard format [http://samtools.sourceforge.net/SAM1.pdf glf format]. Sample files are available at [ftp://share.sph.umich.edu/1000genomes/pilot1/examples/glf.tgz sample glf files].
To get started, you will need glf files in the standard format [http://samtools.sourceforge.net/SAM1.pdf glf format]. Sample files are available at [ftp://share.sph.umich.edu/1000genomes/pilot1/examples/glf.tgz sample glf files].
If you do not have glf files, you can generate them from bam files (bam format also specified in [http://samtools.sourceforge.net/SAM1.pdf glf format bam format]) using the following command line:
If you do not have glf files, you can generate them from bam files (bam format also specified in [http://samtools.sourceforge.net/SAM1.pdf glf format bam format]) using the following command line:
samtools pileup -g -T 1 -f ref.fa my.bam > my.glf
samtools pileup -g -T 1 -f ref.fa my.bam > my.glf
Note: you will need the reference fasta file ref.fa to create glf file from bam file.
Note: you will need the reference fasta file ref.fa to create glf file from bam file.
== How to Run ==
== How to Run ==
This variant calling pipeline has two steps. (step 1) promotion of a set of potential polymorphisms; and (step 2) genotype/haplotype calling using LD information.
This variant calling pipeline has two steps. (step 1) promotion of a set of potential polymorphisms; and (step 2) genotype/haplotype calling using LD information.
(step 1) Site promotion using software glfMultiples [https://www.sph.umich.edu/csg/yli/GPT_Freq.011.source.tgz GPT_Freq].
=== (step 1) Site promotion using software glfMultiples [https://csg.sph.umich.edu//yli/GPT_Freq.011.source.tgz GPT_Freq] ===
GPT_Freq -b my.out -p 0.9 --minDepth 10 --maxDepth 1000 *.glf
GPT_Freq -b my.out -p 0.9 --minDepth 10 --maxDepth 1000 *.glf
(step 2) Genotype/haplotype calling using thunder [https://www.sph.umich.edu/csg/yli/thunder.V009.source.tgz thunder_glf_freq]
minDepth and maxDepth are the cutoffs on total depth (across all individuals). We have found it useful to exclude sites with extremely low and high total depth. Please see Important Filters below.
=== (step 2) Genotype/haplotype calling using thunder [https://csg.sph.umich.edu//yli/thunder/thunder.V011.source.tgz thunder_glf_freq] ===
thunder_glf_freq --shotgun my.out.$chr --detailedInput -r 100 --states 200 --dosage --phase --interim 25 -o my.final.out
Notes:
(1) The program thunder used in step 2 is an extension of MaCH, the genotype imputation software we have previously developed. For details regarding the shared options, please check out [http://sph.umich.edu/csg/abecasis/mach/index.html MaCH website] and [http://genome.sph.umich.edu/wiki/Mach MaCH wiki].
(2) Check out example files and command lines under examples/thunder/ in the thunder package [https://sph.umich.edu/csg/abecasis/thunder/thunder.V011.source.tgz thunder_glf_freq].
== Example Showing the Whole Pipeline ==
In the thunder [https://csg.sph.umich.edu//yli/thunder/thunder.V011.source.tgz thunder_glf_freq] tarball, you can find under example/thunder/ folder, input files extracted from real data and a C-shell script that executes the whole analysis pipeline.
== Ligate Haplotypes ==
Please use [http://csg.sph.umich.edu//yli/ligateHap.V004.tgz ligateHaplotypes].
== Important Filters ==
== Important Filters ==
We have found that the following filters are helpful.
=== allelic imbalance ===
A statistic developed by Dr. Tom Blackwell [http://genome.sph.umich.edu/wiki/Genotype_Likelihood_Based_Allele_Balance allelic imbalance].
=== indel filter ===
We recommend distance to known indels >= 5bp. A catalog of known indels can be found at [ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/pilot_data/release/2010_07/low_coverage/indels/ indel catalog].
=== site promotion filter ===
We recommend setting parameter -p at least >= 0.9 in step 1 (running glfMultiples).
=== strand bias filter ===
=== total depth filter ===
For the 1000 Genomes Project (average depth per individual ~4X), we have found it useful to exclude sites with average total depth per individual < 0.5X or > 20X.
=== coverage filter ===
We recommend the filter of >50% individuals with coverage.
=== flanking sequence filter ===
We recommend excluding sites with >0.1% flanking 10-mer frequency among candidate sites. samtools calmd -br performs this base quality re-calibration.
== Citation ==
Li Y, Sidore C, Kang HM, Boehnke M, Abecasis GR. Low-coverage sequencing: Implications for design of complex trait association studies. <em>Genome Res.</em> 2011 Jun;21(6):940-51. <br>
== Inference with External Reference ==
Please refer to [http://genome.sph.umich.edu/wiki/UMAKE UMAKE]. <br>
== Questions and Comments? ==
Email [mailto:yunli@med.unc.edu Yun Li].
