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| =Brief description= | | = NEW VERSION = |
| This page contains information about the program called NgsRelate, which can be used to infer relatedness coefficients for pairs of individuals from low coverage Next Generation Sequencing (NGS) data by using genotype likelihoods instead of called genotypes. To be able to infer the relatedness you will need to know the population frequencies and have genotype likelihoods. This can be obtained e.g. using the program ANGSD as shown in the examples below. For more information about ANGSD see here: http://popgen.dk/angsd/index.php/Quick_Start.
| | For the NEW version of ngsRelate that coestimates relatedness and inbreeding go to this link https://github.com/ANGSD/NgsRelate |
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| Method is published here: http://bioinformatics.oxfordjournals.org/content/early/2015/08/29/bioinformatics.btv509.abstract
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| =How to download and install= | | = OLD VERSION = |
| The source code for NgsRelate is deposited on github: https://github.com/ANGSD/NgsRelate. On a linux or mac system with curl and g++ installed NgsRelate can be downloaded and installed as follows:
| | For the old version please use this link: http://www.popgen.dk/software/index.php?title=NgsRelate&oldid=694 |
| <pre>
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| curl https://raw.githubusercontent.com/ANGSD/NgsRelate/master/NgsRelate.cpp >NgsRelate.cpp
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| g++ NgsRelate.cpp -O3 -lz -o ngsrelate
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| </pre>
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| =Run examples=
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| Below are two examples of how NgsRelate can be used to estimate relatedness from NGS data. Note that to be able to run all steps of the examples you need to have the programs ANGSD and PLINK installed and you also need to download large data files from both HapMap3 and 1000 Genomes webpages. Furthermore, the examples take several hours to run all in all. They are therefore just meant as illustrations of how NgsRelate can be run. '''If you want to quickly try out NgsRelate, e.g. to check if your installation works, you can download the final input data for NgsRelate used in the very last command in run example 2 here: http://www.popgen.dk/ida/NgsRelateExampleData/web/input/. Using that data you can try out NgsRelate by running that last command, i.e.'''
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| <pre>
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| ./ngsrelate -g angsdput.glf.gz -n 6 -f freq -s 1 >res
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| </pre>
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| The output should be a file called res that contains relatedness estimates for all pairs between 6 individuals. A copy of this file can be found here http://www.popgen.dk/ida/NgsRelateExampleData/web/output/.
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| == Run example 1: using only NGS data==
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| Assume we have file containing paths to 100 BAM/CRAM files; one line per BAN/CRAM file. Then we can use ANGSD to estimate frequencies and calculate genotype likelihoods while doing SNP calling and in the end produce the the input files needed for the NgsRelate program as follows:
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| <pre>
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| ### First we generate a file with allele frequencies (angsdput.mafs.gz) and a file with genotpe likelihoods (angsdput.glf.gz).
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| ./angsd -b filelist -gl 1 -domajorminor 1 -snp_pval 1e-6 - domaf 1 -minmaf 0.05 -doGlf 3
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| ### Then we extract the frequency column from the allele frequency file and remove the header (to make it in the format NgsRelate needs)
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| cut -f5 angsdput.mafs.gz |sed 1d >freq
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| </pre>
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| Once we have these files we can use NgsRelate to estimate relatedness between any pairs of individuals. E.g. if we want to estimate relatedness between the first two individuals (0 and 1) we can do it using the following command:
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| <pre>
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| ./ngsrelate -g angsdput.glf.gz -n 100 -f freq -a 0 -b 1 >gl.res
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| </pre>
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| Here we specify the name of our file with genotype likelihoods after the option "-g", the number of individuals in the file after the option "-n", the name of the file with allele frequencies after the option "-f" and the number of the two individuals after the options "-a" and "-b" . If -a and -b are not specified NgsRelate will loop through all pairs of individuals in the input file.
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| == Run example 2: using NGS data with population frequencies estimated from genetic data from PLINK files ==
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| In this example we show how you can estimate relatedness between a number of individuals which you have NGS data from (in bam files) using genetic data from PLINK files for frequency estimation.
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| Assume the individuals we want to estimate relatedness from are from the population called LWK and assume we have files with genetic data from individuals from LWK as well as other populations in binary PLINK format (e.g. hapmap3_r2_b36_fwd.consensus.qc.polyHg19.*) and a file, LWK.fam, with the IDs of the LWK individuals in this dataset. Then using PLINK we can produce allele frequency information in a format that NgsRelate can use as follows:
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| <pre>
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| ### extract individuals from LWK from huge binary plink file
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| plink --bfile hapmap3_r2_b36_fwd.consensus.qc.polyHg19 --keep LWK.fam --make-bed --out hapmap3Hg19LWK --noweb
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| ### calculate frequencies for this population
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| plink --bfile hapmap3Hg19LWK --freq --noweb --out LWKsub
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| </pre>
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| Afterwards we can use ANGSD to calculate genotype likelihoods for the sites for which we have frequency info for as follows:
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| <pre>
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| ### extract the chr,pos,major,minor information about the sites we have frequency info from into a file
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| ### (so we can extract data from these sites from the NGS data files)
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| cut -f1,4-6 hapmap3Hg19LWK.bim >forAngsd.txt
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| ### index this file for angsd
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| ./angsd sites index forAngsd.txt
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| ### calculate genotype likelihoods for the six individuals for the sites we have frequency info on based on the bam files
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| ### (assuming the paths to the bam files are listed in the file 'list'):
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| ./angsd -gl 1 -doglf 3 -sites forAngsd.txt -b list -domajorminor 3 -P 2 -minMapQ 30 -minQ 20
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| ### this generates the output files angsdput.glf.gz and a angsdput.glf.pos.gz.
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| </pre>
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| Finally we can use NgsRelate to estimate relatedness for the six individuals from which we have NGS data in bam files:
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| <pre>
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| ### extract the frequencies and sync it to the angsd output
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| ./ngsrelate extract_freq angsdput.glf.pos.gz hapmap3Hg19LWK.bim LWKsub.frq >freq
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| ### run ngsrelate
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| ./ngsrelate -g angsdput.glf.gz -n 6 -f freq -s 1 >res
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| </pre>
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| The final relatedness estimates will then be available in the file called "res" which can be found here: http://www.popgen.dk/ida/NgsRelateExampleData/web/output/. Note that we here used the option -s 1 to flip the allele frequencies (i.e. set them to 1 minus the frequencies in the freq file).
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| =Output format= | |
| Example of output of with two samples
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| <pre>
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| Pair k0 k1 k2 loglh nIter coverage
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| (0,1) 0.673213 0.326774 0.000013 -1710940.769941 19 0.814658
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| </pre>
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| Example of output with 6 samples:
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| <pre>
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| cat res
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| Pair k0 k1 k2 loglh nIter coverage
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| (0,1) 0.675337 0.322079 0.002584 -1710946.832375 10 0.813930
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| (0,2) 0.458841 0.526377 0.014782 -1666215.528333 10 0.808822
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| (0,3) 1.000000 0.000000 0.000000 -1743992.363193 -1 0.816266
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| (0,4) 1.000000 0.000000 0.000000 -1759202.971213 -1 0.818856
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| (0,5) 1.000000 0.000000 0.000000 -1550475.615322 -1 0.752663
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| (1,2) 0.007111 0.991020 0.001868 -1580995.130867 10 0.806912
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| (1,3) 1.000000 0.000000 0.000000 -1728859.988212 -1 0.814272
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| (1,4) 1.000001 -0.000001 0.000000 -1744055.203870 9 0.816887
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| (1,5) 1.000000 0.000000 0.000000 -1536858.187440 -1 0.750917
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| (2,3) 1.000000 0.000000 0.000000 -1705157.832621 -1 0.809297
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| (2,4) 1.000000 0.000000 0.000000 -1719681.338365 -1 0.811804
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| (2,5) 1.000000 0.000000 0.000000 -1517388.260612 -1 0.746903
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| (3,4) 0.547602 0.439423 0.012975 -1743899.789842 10 0.819276
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| (3,5) 0.265819 0.482953 0.251228 -1467343.087647 10 0.754637
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| (4,5) 0.004655 0.995345 -0.000000 -1473415.049411 8 0.755734
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| </pre>
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| The first column contains the information of about which individuals was used for the analysis. The next three columns are the maximum likelihood (ML) estimate of the relatedness coefficients. The fifth column is the log of the likelihood of the ML estimate. The sixth column is the number of iterations of the maximization algorithm that was used to find the MLE, and finally the seventh column is fraction of non-missing sites, i.e. the fraction of sites where data was available for both individuals, and where the minor allele frequency (MAF) above the threshold (default is 0.05 but the user may specify a different threshold). Note that in some cases nIter is -1. This indicates that values on the boundary of the parameter space had a higher likelihood than the values achieved using the EM-algorithm (ML methods sometimes have trouble finding the ML estimate when it is on the boundary of the parameter space, and we therefore test the boundary values explicitly and output these if these have the highest likelihood).
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| = Input file format =
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| NgsAdmix takes two files as input: a file with genotype likelihoods and a file with frequencies for the sites there are genotype likelihoods for.
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| The genotype likelihood file needs to contain a line for each site with 3 values for each individual (one log transformed genotype likelihood for each of the 3 possible genotypes encoded as 'double's) and it needs to be in binary format and gz compressed.
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| The frequency file needs to contain a line per site with the allele frequency of the site in it.
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| = Help and additional options =
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| To get help and a list of all options simply type
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| <pre>
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| ./ngsrelate
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| </pre>
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| = Citing and references =
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| = Changelog =
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| See github for log
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| =Bugs/Improvements=
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| -Make better output message if files doesn't exists when using the extract_freq option
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