Population variant calling pipeline - Revision history

Dawes0011 at 08:59, 16 June 2023

2023-06-16T08:59:21Z

← Older revision		Revision as of 10:59, 16 June 2023
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	Check if the cache file for your species already exist in <code>/lustre/nobackup/SHARED/cache/</code>. If it doesn't, create it with		Check if the cache file for your species already exist in <code>/lustre/nobackup/SHARED/cache/</code>. If it doesn't, create it with

	<pre>/usr/bin/perl ~~/cm~~/shared/apps/SHARED/ensembl-vep/INSTALL.pl --CACHEDIR /lustre/nobackup/SHARED/cache/ --AUTO c -n --SPECIES <species></pre>		<pre>/usr/bin/perl /shared/apps/SHARED/ensembl-vep/INSTALL.pl --CACHEDIR /lustre/nobackup/SHARED/cache/ --AUTO c -n --SPECIES <species></pre>
	When multiple assemblies are found you need to run it again with <code>--ASSEMBLY <assembly name></code>, where "assembly name" is the name of the assembly you want to use.		When multiple assemblies are found you need to run it again with <code>--ASSEMBLY <assembly name></code>, where "assembly name" is the name of the assembly you want to use.

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	When multiple assemblies are found you need to run it again with <code>--ASSEMBLY <assembly name></code>, where "assembly name" is the name of the assembly you want to use.		When multiple assemblies are found you need to run it again with <code>--ASSEMBLY <assembly name></code>, where "assembly name" is the name of the assembly you want to use.

	In the Snakefile, in rule <code>run_vep</code>, replace <code>~~/cm~~/shared/apps/SHARED/ensembl-vep/vep</code> with <code>vep</code>		In the Snakefile, in rule <code>run_vep</code>, replace <code>/shared/apps/SHARED/ensembl-vep/vep</code> with <code>vep</code>

	<span id="installing-r-packages"></span>		<span id="installing-r-packages"></span>
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	<pre>Warning in install.packages(new.packages) :		<pre>Warning in install.packages(new.packages) :
	'lib = "~~/cm~~/shared/apps/R/3.6.2/lib64/R/library"' is not writable</pre>		'lib = "/shared/apps/R/3.6.2/lib64/R/library"' is not writable</pre>
	Follow the instructions on how to install R packages locally [https://wiki.anunna.wur.nl/index.php/Installing_R_packages_locally here] and try to install the packages again.		Follow the instructions on how to install R packages locally [https://wiki.anunna.wur.nl/index.php/Installing_R_packages_locally here] and try to install the packages again.

Moiti001: added author info

2022-06-13T14:34:13Z

added author info

← Older revision		Revision as of 16:34, 13 June 2022
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			Author: Carolina Pita Barros <br />
			Contact: carolina.pitabarros@wur.nl <br />
			ABG<br />

			For up-to-date documentation see [https://github.com/CarolinaPB/population-variant-calling here]

	= Population level variant calling =		= Population level variant calling =

Moiti001: added info on how to configure VEP and install R packages

2022-03-02T14:18:57Z

added info on how to configure VEP and install R packages

@@ Line 56: / Line 56: @@
 * SPECIES - species name to be used for VEP
 * NUM_CHRS - number of chromosomes for your species (necessary for plink). ex: 38
 <span id="results"></span>

Moiti001: added workflow image

2022-03-02T13:45:28Z

added workflow image

← Older revision		Revision as of 15:45, 2 March 2022
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	{\|		{\|
	!align="center"\| [[File:~~https://github.com/CarolinaPB/pop~~-var-calling~~/blob/master/~~workflow.png~~\|DAG~~]]		!align="center"\| [[File:population-var-calling-workflow.png]]
	\|-		\|-
	\|align="center"\| ''Pipeline workflow''		\|align="center"\| ''Pipeline workflow''

Moiti001: added info on pipeline

2022-03-02T13:43:18Z

added info on pipeline

New page

= Population level variant calling =

Path to pipeline: /lustre/nobackup/WUR/ABGC/shared/PIPELINES/population-variant-calling


== First follow the instructions here ==

[https://carolinapb.github.io/2021-06-23-how-to-run-my-pipelines/ Step by step guide on how to use my pipelines] 
Click [https://github.com/CarolinaPB/snakemake-template/blob/master/Short%20introduction%20to%20Snakemake.pdf here] for an introduction to Snakemake


== ABOUT ==

This is a pipeline that takes short reads aligned to a genome (in <code>.bam</code> format) and performs population level variant calling with <code>Freebayes</code>. It uses VEP to annotate the resulting VCF, calculates statistics, and calculates and plots a PCA.

It was developed to work with the results of [https://github.com/CarolinaPB/population-mapping this population mapping pipeline]. There are a few <code>Freebayes</code> requirements that you need to take into account if you don't use the mapping pipeline mentioned above to map your reads. You should make sure that:

* Alignments have read groups
* Alignments are sorted
* Duplicates are marked

See [https://github.com/freebayes/freebayes#calling-variants-from-fastq-to-vcf here] for more details.


==== Tools used ====

* [https://github.com/freebayes/freebayes Freebayes] - variant calling using short reads
* [https://samtools.github.io/bcftools/bcftools.html bcftools] - vcf statistics
* [https://www.cog-genomics.org/plink/ Plink] - compute PCA
* R - Plot PCA

{|
!align="center"| [[File:https://github.com/CarolinaPB/pop-var-calling/blob/master/workflow.png|DAG]]
|-
|align="center"| ''Pipeline workflow''
|}


=== Edit config.yaml with the paths to your files ===

<syntaxhighlight lang="yaml">ASSEMBLY: /path/to/fasta
MAPPING_DIR: /path/to/bams/dir
PREFIX: <prefix>
OUTDIR: /path/to/outdir
SPECIES: <species>
NUM_CHRS: <number of chromosomes></syntaxhighlight>
* ASSEMBLY - path to genome fasta file
* MAPPING_DIR - path to directory with bam files to be used
** the pipeline will use all bam files in the directory, if you want to use a subset of those, create a file named <code>bam_list.txt</code> that contains the paths to the bam files you want to use. One path per line.

<syntaxhighlight lang="text">/path/to/file.bam
/path/to/file2.bam</syntaxhighlight>
* PREFIX - prefix for the created files
* OUTDIR - directory where snakemake will run and where the results will be written to 
If you want the results to be written to this directory (not to a new directory), open config.yaml and comment out <code>OUTDIR: /path/to/outdir</code>
* SPECIES - species name to be used for VEP
* NUM_CHRS - number of chromosomes for your species (necessary for plink). ex: 38


== RESULTS ==

The most important files and directories are:

* '''<run_date>_files.txt''' dated file with an overview of the files used to run the pipeline (for documentation purposes)
* '''results''' directory that contains
** '''final_VCF''' directory with variant calling VCF files, as well as VCF stats
*** {prefix}.vep.vcf.gz - final VCF file
*** {prefix}.vep.vcf.gz.stats
** '''PCA''' PCA results and plot
*** {prefix}.eigenvec and {prefix}.eigenval - file with PCA eigenvectors and eigenvalues, respectively
*** {prefix}.pdf - PCA plot

The VCF file has been filtered for <code>QUAL > 20</code>. Freebayes is ran with parameters <code>--use-best-n-alleles 4 --min-base-quality 10 --min-alternate-fraction 0.2 --haplotype-length 0 --ploidy 2 --min-alternate-count 2</code>. These parameters can be changed in the Snakefile.