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.

Quick Start: Difference between revisions

From angsd
Jump to navigation Jump to search
No edit summary
 
(37 intermediate revisions by 2 users not shown)
Line 1: Line 1:
This page contains some random examples that shows some aspect of the ANGSD program. There is also an old [[Tutorial]], but this is somewhat outdated. We assume you will have SAMtools installed.
This page all contain some random examples that shows some aspect of the ANGSD program. We assume you will have SAMtools installed for general BAM/CRAM manipulation


Many of the examples in the individual subpages are based on this test data set. The examples below are just some random examples.
Many of the examples in the individual subpages are based on this test data set. The examples below are just some random examples.
=Download and prepare=
=Download and prepare=
==BAM files from 1000genomes project==
First download some test data of random small BAM files which contains some regions from different chromosomes for 10 samples from the 1000genomes project. The file size is around 100megabytes.
First download some test data of random small BAM files which contains some regions from different chromosomes for 10 samples from the 1000genomes project. The file size is around 100megabytes.
<pre>
<pre>
Line 9: Line 10:
</pre>
</pre>
This has made a folder called '''bams/''', which contains our 10 samples.  
This has made a folder called '''bams/''', which contains our 10 samples.  
Now download and install angsd you can follow the guidelines at the [[Installation]] page which basicly says:
Now download and install angsd you can follow the guidelines at the [[Installation]] page.
<pre>
 
wget http://popgen.dk/software/download/angsd/angsd0.570.tar.gz
tar xfz angsd0.570.tar.gz
cd angsd0.570
make
cd ..
</pre>
We will also index the BAM files in case we need to do random access, for this we will use SAMtools.
We will also index the BAM files in case we need to do random access, for this we will use SAMtools.
<pre>
<pre>
Line 26: Line 21:
ls bams/*.bam > bam.filelist
ls bams/*.bam > bam.filelist
</pre>
</pre>
==Ancestral fasta file for hg19==
If you want to run the SFS examples on the wiki you should also download the ancestral states for the hg19 assembly of the human genome.
<pre>
wget http://popgen.dk/software/download/angsd/hg19ancNoChr.fa.gz
mv hg19ancNoChr.fa.gz chimpHg19.fa.gz
samtools faidx chimpHg19.fa.gz
</pre>
NB some have complained that the popgen.dk server is unstable, and we are therefore also hosting the compressed .fa file here:
http://dna.ku.dk/~thorfinn/hg19ancNoChr.fa.gz


=Examples=
=Examples=
Line 32: Line 37:


<div class="toccolours mw-collapsible mw-collapsed">
<div class="toccolours mw-collapsible mw-collapsed">
./angsd0.570/angsd -b list -GL 1 -doMajorMinor 1 -doMaf 2 -P 5  
./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5  
<pre class="mw-collapsible-content">
<pre class="mw-collapsible-content">
Command:
Command:
./angsd0.574/angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5  
./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5
-> angsd version: 0.574 build(Jan 10 2014 17:44:41)
-> angsd version: 0.574 build(Jan 10 2014 17:44:41)
-> No '-out' argument given, output files will be called 'angsdput'
-> No '-out' argument given, output files will be called 'angsdput'
Line 54: Line 59:
</div>
</div>


The output is then located on angsdput.mafs.gz. We could have specified an output file name with '''-out'''. Lets remove those reads that has a mapping quality below 30, and only use the bases with a score above 19. And to simply output we only print those sites with an allele frequency above 0.05.
The output is then located on angsdput.mafs.gz. We could have specified an different output file name with '''-out'''. Lets remove those reads that has a mapping quality below 30, and only use the bases with a score above 19. And to simply output we only print those sites with an allele frequency above 0.05.


<div class="toccolours mw-collapsible mw-collapsed">
<div class="toccolours mw-collapsible mw-collapsed">
./angsd0.574/angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5 -minMapQ 30 -minBaseQ 20 -minMaf 0.05
./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5 -minMapQ 30 -minQ 20 -minMaf 0.05
<pre class="mw-collapsible-content">
<pre class="mw-collapsible-content">
Command:
Command:
./angsd0.574/angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5 -minMapQ 30 -minQ 20 -minMaf 0.05  
./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5 -minMapQ 30 -minQ 20 -minMaf 0.05  
-> angsd version: 0.574 build(Jan 10 2014 17:44:41)
-> angsd version: 0.574 build(Jan 10 2014 17:44:41)
-> No '-out' argument given, output files will be called 'angsdput'
-> No '-out' argument given, output files will be called 'angsdput'
Line 77: Line 82:
</pre>
</pre>
</div>
</div>
=adf=
And lets look at the output:
And lets look at the output:
<div class="toccolours mw-collapsible mw-collapsed">
<div class="toccolours mw-collapsible mw-collapsed">
asdf
gunzip -c angsdput.mafs.gz |head
<pre class="mw-collapsible-content">
<pre class="mw-collapsible-content">
asdf
chromo position major minor unknownEM nInd
1 14000023 C A 0.076211 4
1 14000176 G A 0.117885 6
1 14000202 G A 0.052565 6
1 14000873 G A 0.295131 9
1 14001018 T C 0.269244 9
1 14001202 G T 0.065666 8
1 14001501 G A 0.062746 10
1 14001867 A G 0.272099 10
1 14002093 T C 0.058891 10
</pre>
</pre>


</div>
</div>


asdfasdf
We have 10 samples, lets only look at the sites where we have information from at least 8 individuals.
<div class="toccolours mw-collapsible mw-collapsed">
<div class="toccolours mw-collapsible mw-collapsed">
asdf
./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5 -minMapQ 30 -minQ 20 -minMaf 0.05 -minInd 8
<pre class="mw-collapsible-content">
<pre class="mw-collapsible-content">
asdf
Command:
./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5 -minMapQ 30 -minQ 20 -minMaf 0.05 -minInd 8
-> angsd version: 0.574 build(Jan 10 2014 17:44:41)
-> No '-out' argument given, output files will be called 'angsdput'
-> Parsing 10 number of samples
-> Printing at chr: 20 pos:14085533 chunknumber 2800
-> Done reading data waiting for calculations to finish
-> Calling destroy
-> Done waiting for threads
-> Output filenames:
->"angsdput.arg"
->"angsdput.mafs.gz"
-> Fri Jan 10 18:11:09 2014
-> Arguments and parameters for all analysis are located in .arg file
[ALL done] cpu-time used =  36.64 sec
[ALL done] walltime used =  30.00 sec
 
</pre>
</pre>


</div>
</div>
And look at the output:
<div class="toccolours mw-collapsible mw-collapsed">
gunzip -c angsdput.mafs.gz |head
<pre class="mw-collapsible-content">
chromo position major minor unknownEM nInd
1 14000873 G A 0.295131 9
1 14001018 T C 0.269244 9
1 14001202 G T 0.065666 8
1 14001501 G A 0.062746 10
1 14001867 A G 0.272099 10
1 14002093 T C 0.058891 10
1 14002342 C T 0.078385 9
1 14002422 A T 0.434411 9
1 14002474 T C 0.072898 8
</pre>
</div>
==Generate Beagle Likelihood files==
For diploid samples we have 10 possible genotypes. but we would only expect to observe two different alleles at most sites. We can infer the major and minor allele and output the 3 possible genotypes in beagle genotype likelihood format. Furthermore we are only interested in variable sites, so let us use the LRT statistic for filtering out the sites that are very likely to be polymorphic with a p-value less than 10^-6. We need to allele frequency in order to test if a site is polymorphic.
<div class="toccolours mw-collapsible mw-collapsed">
./angsd -GL 2 -doGlf 2 -b bam.filelist -doMajorMinor 1 -SNP_pval 1e-6 -doMaf 1
<pre class="mw-collapsible-content">
Command:
./angsd -GL 2 -doGlf 2 -b bam.filelist -doMajorMinor 1 -SNP_pval 1e-6 -doMaf 1
-> angsd version: 0.574 build(Jan 10 2014 17:44:41)
-> No '-out' argument given, output files will be called 'angsdput'
-> Parsing 10 number of samples
-> Printing at chr: 20 pos:14095816 chunknumber 3500
-> Done reading data waiting for calculations to finish
-> Calling destroy
-> Done waiting for threads
-> Output filenames:
->"angsdput.arg"
->"angsdput.beagle.gz"
-> Fri Jan 10 18:26:56 2014
-> Arguments and parameters for all analysis are located in .arg file
[ALL done] cpu-time used =  27.24 sec
[ALL done] walltime used =  27.00 sec
</pre>
</div>
Let us look at the generated output file: angsdput.beagle.gz. We only look at the data for the first 2 individuals.
<div class="toccolours mw-collapsible mw-collapsed">
gunzip -c angsdput.beagle.gz | less -S
<pre class="mw-collapsible-content">
marker allele1 allele2 Ind0 Ind0 Ind0 Ind1 Ind1 Ind1
1_14000202 2 0 0.000532 0.999468 0.000000 0.333333 0.333333 0.333333
1_14000873 2 0 0.000000 0.030324 0.969676 0.663107 0.333333 0.003560
1_14001018 3 1 0.000000 0.015429 0.984571 0.799823 0.200177 0.000000
1_14001867 0 2 0.000056 0.333333 0.666611 0.888793 0.111207 0.000000
1_14002342 1 3 0.941072 0.058928 0.000000 0.888806 0.111194 0.000000
1_14002422 0 3 0.000000 0.111147 0.888853 0.799777 0.200223 0.000000
1_14002474 3 1 0.969662 0.030338 0.000000 0.799810 0.200190 0.000000
1_14003581 1 3 0.000000 0.200027 0.799973 0.984577 0.015423 0.000000
1_14004623 3 1 0.000000 0.200035 0.799965 0.984501 0.015499 0.000000
</pre>
</div>
Notice that the 'marker' contains the genomic position, encoded by using underscore as separator.

Latest revision as of 13:58, 4 December 2015

This page all contain some random examples that shows some aspect of the ANGSD program. We assume you will have SAMtools installed for general BAM/CRAM manipulation

Many of the examples in the individual subpages are based on this test data set. The examples below are just some random examples.

Download and prepare

BAM files from 1000genomes project

First download some test data of random small BAM files which contains some regions from different chromosomes for 10 samples from the 1000genomes project. The file size is around 100megabytes.

wget http://popgen.dk/software/download/angsd/bams.tar.gz
tar xf bams.tar.gz

This has made a folder called bams/, which contains our 10 samples. Now download and install angsd you can follow the guidelines at the Installation page.

We will also index the BAM files in case we need to do random access, for this we will use SAMtools.

for i in bams/*.bam;do samtools index $i;done

We make a file containing a list of the locations of the 10 bamfiles

ls bams/*.bam > bam.filelist

Ancestral fasta file for hg19

If you want to run the SFS examples on the wiki you should also download the ancestral states for the hg19 assembly of the human genome.

wget http://popgen.dk/software/download/angsd/hg19ancNoChr.fa.gz
mv hg19ancNoChr.fa.gz chimpHg19.fa.gz
samtools faidx chimpHg19.fa.gz

NB some have complained that the popgen.dk server is unstable, and we are therefore also hosting the compressed .fa file here: http://dna.ku.dk/~thorfinn/hg19ancNoChr.fa.gz

Examples

Calculate Allele frequencies

Assuming you have a list of bamfiles in in file: 'bam.filelist' and you want the MAF using all reads and inferring the major and minor from the GL, we will use SAMtools genotype likelihoods, and will allow for 5 threads: See details on Allele Frequencies, Major Minor and Genotype Likelihoods.

./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5

	Command:
./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5
	-> angsd version: 0.574	 build(Jan 10 2014 17:44:41)
	-> No '-out' argument given, output files will be called 'angsdput'
	-> Parsing 10 number of samples 
	-> Printing at chr: 20 pos:14095816 chunknumber 3500
	-> Done reading data waiting for calculations to finish
	-> Calling destroy
	-> Done waiting for threads
	-> Output filenames:
		->"angsdput.arg"
		->"angsdput.mafs.gz"
	-> Fri Jan 10 17:46:15 2014
	-> Arguments and parameters for all analysis are located in .arg file
	[ALL done] cpu-time used =  130.67 sec
	[ALL done] walltime used =  55.00 sec

The output is then located on angsdput.mafs.gz. We could have specified an different output file name with -out. Lets remove those reads that has a mapping quality below 30, and only use the bases with a score above 19. And to simply output we only print those sites with an allele frequency above 0.05.

./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5 -minMapQ 30 -minQ 20 -minMaf 0.05

Command:
./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5 -minMapQ 30 -minQ 20 -minMaf 0.05 
	-> angsd version: 0.574	 build(Jan 10 2014 17:44:41)
	-> No '-out' argument given, output files will be called 'angsdput'
	-> Parsing 10 number of samples 
	-> Printing at chr: 20 pos:14085533 chunknumber 2800
	-> Done reading data waiting for calculations to finish
	-> Calling destroy
	-> Done waiting for threads
	-> Output filenames:
		->"angsdput.arg"
		->"angsdput.mafs.gz"
	-> Fri Jan 10 17:57:55 2014
	-> Arguments and parameters for all analysis are located in .arg file
	[ALL done] cpu-time used =  123.48 sec
	[ALL done] walltime used =  51.00 sec

And lets look at the output:

gunzip -c angsdput.mafs.gz |head

chromo	position	major	minor	unknownEM	nInd
1	14000023	C	A	0.076211	4
1	14000176	G	A	0.117885	6
1	14000202	G	A	0.052565	6
1	14000873	G	A	0.295131	9
1	14001018	T	C	0.269244	9
1	14001202	G	T	0.065666	8
1	14001501	G	A	0.062746	10
1	14001867	A	G	0.272099	10
1	14002093	T	C	0.058891	10

We have 10 samples, lets only look at the sites where we have information from at least 8 individuals.

./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5 -minMapQ 30 -minQ 20 -minMaf 0.05 -minInd 8

Command:
./angsd -b bam.filelist -GL 1 -doMajorMinor 1 -doMaf 2 -P 5 -minMapQ 30 -minQ 20 -minMaf 0.05 -minInd 8
	-> angsd version: 0.574	 build(Jan 10 2014 17:44:41)
	-> No '-out' argument given, output files will be called 'angsdput'
	-> Parsing 10 number of samples 
	-> Printing at chr: 20 pos:14085533 chunknumber 2800
	-> Done reading data waiting for calculations to finish
	-> Calling destroy
	-> Done waiting for threads
	-> Output filenames:
		->"angsdput.arg"
		->"angsdput.mafs.gz"
	-> Fri Jan 10 18:11:09 2014
	-> Arguments and parameters for all analysis are located in .arg file
	[ALL done] cpu-time used =  36.64 sec
	[ALL done] walltime used =  30.00 sec

And look at the output:

gunzip -c angsdput.mafs.gz |head

chromo	position	major	minor	unknownEM	nInd
1	14000873	G	A	0.295131	9
1	14001018	T	C	0.269244	9
1	14001202	G	T	0.065666	8
1	14001501	G	A	0.062746	10
1	14001867	A	G	0.272099	10
1	14002093	T	C	0.058891	10
1	14002342	C	T	0.078385	9
1	14002422	A	T	0.434411	9
1	14002474	T	C	0.072898	8

Generate Beagle Likelihood files

For diploid samples we have 10 possible genotypes. but we would only expect to observe two different alleles at most sites. We can infer the major and minor allele and output the 3 possible genotypes in beagle genotype likelihood format. Furthermore we are only interested in variable sites, so let us use the LRT statistic for filtering out the sites that are very likely to be polymorphic with a p-value less than 10^-6. We need to allele frequency in order to test if a site is polymorphic.

./angsd -GL 2 -doGlf 2 -b bam.filelist -doMajorMinor 1 -SNP_pval 1e-6 -doMaf 1

Command:
./angsd -GL 2 -doGlf 2 -b bam.filelist -doMajorMinor 1 -SNP_pval 1e-6 -doMaf 1 
	-> angsd version: 0.574	 build(Jan 10 2014 17:44:41)
	-> No '-out' argument given, output files will be called 'angsdput'
	-> Parsing 10 number of samples 
	-> Printing at chr: 20 pos:14095816 chunknumber 3500
	-> Done reading data waiting for calculations to finish
	-> Calling destroy
	-> Done waiting for threads
	-> Output filenames:
		->"angsdput.arg"
		->"angsdput.beagle.gz"
	-> Fri Jan 10 18:26:56 2014
	-> Arguments and parameters for all analysis are located in .arg file
	[ALL done] cpu-time used =  27.24 sec
	[ALL done] walltime used =  27.00 sec

Let us look at the generated output file: angsdput.beagle.gz. We only look at the data for the first 2 individuals.

gunzip -c angsdput.beagle.gz | less -S

marker allele1 allele2 Ind0 Ind0 Ind0 Ind1 Ind1 Ind1
1_14000202 2 0 0.000532 0.999468 0.000000 0.333333 0.333333 0.333333
1_14000873 2 0 0.000000 0.030324 0.969676 0.663107 0.333333 0.003560
1_14001018 3 1 0.000000 0.015429 0.984571 0.799823 0.200177 0.000000
1_14001867 0 2 0.000056 0.333333 0.666611 0.888793 0.111207 0.000000
1_14002342 1 3 0.941072 0.058928 0.000000 0.888806 0.111194 0.000000
1_14002422 0 3 0.000000 0.111147 0.888853 0.799777 0.200223 0.000000
1_14002474 3 1 0.969662 0.030338 0.000000 0.799810 0.200190 0.000000
1_14003581 1 3 0.000000 0.200027 0.799973 0.984577 0.015423 0.000000
1_14004623 3 1 0.000000 0.200035 0.799965 0.984501 0.015499 0.000000

Notice that the 'marker' contains the genomic position, encoded by using underscore as separator.