ANGSD: Analysis of next generation Sequencing Data
Latest tar.gz version is (0.938/0.939 on github), see Change_log for changes, and download it here.
Contamination
Angsd can estimate contamination, but only for chromosomes that exists in one genecopy (eg chrX for males). This method requires a list of polymorphic sites along with their frequency and we also recommend to discard regions with low mappability.
We have included a mappability and HapMap files for chrX these are found in the RES subfolder of the angsd source package (using hg19). So if you are working with humans, and your sample is a male then you can estimate the contamination with the follow two commands.
- First we generate a binary count file for chrX for a single BAM file (ANGSD cprogram)
- Then we do a Fisher's exact test for finding a p-value, and jackknife to get an estimate of contamination (Rprogram)
An example are found below:
#run angsd ./angsd -i my.bam -r X:5000000-154900000 -doCounts 1 -iCounts 1 -minMapQ 30 -minQ 20 #do jackKnife in R Rscript R/contamination.R mapFile="RES/map100.chrX.gz" hapFile="RES/hapMapCeuXlift.map.gz" countFile="angsdput.icnts.gz" mc.cores=24 #or use the command below for a newer version of the resourcefiles. Rscript R/contamination.R mapFile="RES/chrX.unique.gz" hapFile="RES/HapMapChrX.gz" countFile="angsdput.icnts.gz" mc.cores=24 ##or the fancy fast new c++ program misc/contamination -a angsdput.icnts.gz -h RES/HapMapChrX.gz
The contamination.R program is found in the R/ subfolder, and the resource files are found in the RES folder. The jackknive procedure can be quite slow, so we allocate 24 cores for this analysis mc.cores=24.
The output from the above command is shown below
R
Rscript R/contamination.R Loading required package: parallel -> Needed arguments: hapFile : HapMapfile countFile : Count file -> Optional arguments (defaults): mapFile ( NA ) : Mappability file minDepth ( 2 ) : Minimum depth maxDepth ( 20 ) : Maximium depth mc.cores ( 10 ) : Number of cores fixed ( TRUE ) : Use fixed version of likelihood jack ( TRUE ) : Jacknive to get confidence intervals minmaf ( 0.05 ) : minimum maf startPos ( 5e+06 ) : start position stopPos ( 154900000 ) : stop position seed ( NA ) : set a seed (supply int value)
Rscript ../R/contamination.R mapFile="map100.chrX.bz2" hapFile="hapMapCeuXlift.map.bz2" countFile="angsdput.icnts.gz" mc.cores=24
Loading required package: parallel
-----------------------
Doing Fisher exact test for Method1:
SNP site adjacent site
minor base 616 3554
major base 198492 1589087
Fisher's Exact Test for Count Data
data: mat
p-value = 5.286e-13
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
1.271632 1.512213
sample estimates:
odds ratio
1.387606
-----------------------
Doing Fisher exact test for Method2:
SNP site adjacent site
minor base 114 654
major base 37983 304122
Fisher's Exact Test for Count Data
data: mat2
p-value = 0.001532
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
1.133367 1.705751
sample estimates:
odds ratio
1.395672
-----------------------
major and minor bases - Method1:
-4 -3 -2 -1 SNP site 1 2 3 4
minor base 427 417 475 437 616 486 439 427 446
major base 198651 198715 198656 198645 198492 198500 198681 198693 198546
-----------------------
major and minor bases - Method2:
-4 -3 -2 -1 SNP site 1 2 3 4
minor base 75 76 96 73 114 86 79 80 89
major base 38022 38021 38001 38024 37983 38011 38018 38017 38008
----------------------
Running jackknife for Method1 (could be slow)
Running jackknife for Method2 (could be slow)
$est
Method1 Method2
Contamination 0.03837625 0.03380983
llh 1034.078 483.5145
SE 0.002630455 0.003900376
Interpretation of output
Both methods shows a highly significant pvalue, and estimate the level of contamination to be approx 3%.
C
Later versions of angsd gives a much nice output format. Below is based on a different file from above.
misc/contamination -> Must supply -h hapmapfile -a angsd.icnts.gz file -> Other options: -m minaf -b startpos -c stoppos -d mindepth -e maxdepth -f skiptrans -p nthreads -s seed -j maxjackknife
misc/contamination -a angsdput.icnts.gz -h RES/HapMapChrX.gz -p 100 -s 100 ----------------- hapmap:RES/HapMapChrX.gz counts:angsdput.icnts.gz minMaf:0.050000 startPos:5000000 stopPos:154900000 minDepth:2 maxDepth:200 skiptrans:0 nthreads:100 seed:100 Method2 is subject to fluctuations due to random sampling Seed value of 0 (zero) will use time as seed ----------------- [readhap] We now have: 58190 snpSites after filtering based on hapMapfile [readicnts] fname:angsdput.icnts.gz minDepth:2 maxDepth:200 [readicnts] Has read:70325301 sites, 28883428 sites (after depfilter) from ANGSD icnts file After removing SNP sites with no data in 5bp surrounding region` We have nSNP sites: 9809, with flanking: 88281 -------- MAIN RESULTS: Fisher exact test: Method n11 n12 n21 n22 prob left right twotail method1 316 882 25792 207906 4.60682e-49 1 7.02673e-49 9.33613e-49 method2 107 318 9702 78154 2.18987e-16 1 3.43772e-16 4.01568e-16 Mismatch_rate_for_flanking:0.004224 MisMatch_rate_for_snpsite:0.012104 Method1: old_llh Version: MoM:0.020657 SE(MoM):1.898369e-03 ML:0.020983 SE(ML):1.847108e-03 Method1: new_llh Version: MoM:0.020538 SE(MoM):1.908704e-03 ML:0.020909 SE(ML):1.839107e-03 Method2: old_llh Version: MoM:0.018192 SE(MoM):2.877245e-03 ML:0.018908 SE(ML):2.799355e-03 Method2: new_llh Version: MoM:0.018087 SE(MoM):2.892190e-03 ML:0.018832 SE(ML):2.787229e-03
Method
The method is described in the supplementary of Rasmussen2011