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SeqShop: Variant Calling and Filtering for SNPs Practical, June 2014 (view source)

Revision as of 14:37, 13 November 2014

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'''Note:''' the latest version of this practical is available at: [[SeqShop: Variant Calling and Filtering for SNPs Practical]]
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* The ones here is the original one from the June workshop (updated to be run from elsewhere)
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==Introduction==
 
==Introduction==
 
See the [[Media:SeqShop - GotCloud snpcall.pdf|introductory slides]] for an intro to this tutorial.
 
See the [[Media:SeqShop - GotCloud snpcall.pdf|introductory slides]] for an intro to this tutorial.
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<div class="mw-collapsible-content">
 
<div class="mw-collapsible-content">
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This tutorial builds on the alignment tutorial, if you have not already, please first run that tutorial: [[SeqShop:_Sequence_Mapping_and_Assembly_Practical|Alignment Tutorial]]
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This tutorial builds on the alignment tutorial, if you have not already, please first run that tutorial: [[SeqShop:_Sequence_Mapping_and_Assembly_Practical, June 2014|Alignment Tutorial]]
    
{{SeqShopRemoteEnv}}
 
{{SeqShopRemoteEnv}}
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Per sample BAM files contain sequence reads that are mapped to positions in the genome.
 
Per sample BAM files contain sequence reads that are mapped to positions in the genome.
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For a reminder on how to look at/read BAM files, see: [[SeqShop:_Sequence_Mapping_and_Assembly_Practical#BAM_Files|SeqShop Aligment: BAM Files]]
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For a reminder on how to look at/read BAM files, see: [[SeqShop:_Sequence_Mapping_and_Assembly_Practical, June 2014#BAM_Files|SeqShop Aligment: BAM Files]]
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For this tutorial, we will use the 4 BAMs produced in the [[SeqShop: Sequence Mapping and Assembly Practical]] as well as with 58 BAMs that were pre-aligned to that 1MB region of chromosome 22.
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For this tutorial, we will use the 4 BAMs produced in the [[SeqShop: Sequence Mapping and Assembly Practical, June 2014]] as well as with 58 BAMs that were pre-aligned to that 1MB region of chromosome 22.
    
=== Reference Files ===
 
=== Reference Files ===
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<li>View Screenshot</li>
 
<li>View Screenshot</li>
 
<div class="mw-collapsible-content">
 
<div class="mw-collapsible-content">
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[[File:RefDir.png|500px]]
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[[File:RefDir.png|700px]]
 
</div>
 
</div>
 
</div>
 
</div>
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;Do you notice a difference between this index and yours?
 
;Do you notice a difference between this index and yours?
 
<ul>
 
<ul>
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<div class="mw-collapsible mw-collapsed" style="width:500px">
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<div class="mw-collapsible mw-collapsed" style="width:550px">
 
<li>Answer:</li>
 
<li>Answer:</li>
 
<div class="mw-collapsible-content">
 
<div class="mw-collapsible-content">
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<li>It doesn't have a full path to the BAM file, while your index has /home/...</li>
 
<li>It doesn't have a full path to the BAM file, while your index has /home/...</li>
 
[[File:Bamindex1.png|300px]]
 
[[File:Bamindex1.png|300px]]
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<li>That's ok, <code>gotcloud.conf</code> contains the path to those BAMs</li>
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<li>That's ok, we will use the <code>--base_prefix ${SS}</code> command-line option to prefix the BAM paths</li>
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[[File:BamindexConf.png|300px]]
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<li>Alternatively, we could have set BAM_INDEX in <code>gotcloud.conf</code> to the path to the BAMs
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<pre>BAM_INDEX = /home/username/seqshop/example</pre> </li>
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<ul>
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<li>NOTE: the conf file can't interpret ${SS} environment variables or '~', so you would have to specify the full path</li>
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<li>We just used the command-line option for this tutorial since this path will vary by user.</li>
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</ul>
 
</div>
 
</div>
 
</div>
 
</div>
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We need to add these BAMs to our index
 
We need to add these BAMs to our index
 
* Append the bam.index from the pre-aligned BAMs to the one you generated from the alignment pipeline
 
* Append the bam.index from the pre-aligned BAMs to the one you generated from the alignment pipeline
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** '''Be sure to do this command just once'''
 
  cat ${SS}/bams/bam.index >> ${OUT}/bam.index
 
  cat ${SS}/bams/bam.index >> ${OUT}/bam.index
 
* ">>" will append to the file that follows it
 
* ">>" will append to the file that follows it
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* '''Be sure to do this command just once'''
   
** Check that your BAM index is the correct size
 
** Check that your BAM index is the correct size
 
**:<pre>wc -l ${OUT}/bam.index</pre>
 
**:<pre>wc -l ${OUT}/bam.index</pre>
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We will use the same configuration file as we used yesterday in GotCloud Align.
 
We will use the same configuration file as we used yesterday in GotCloud Align.
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See [[SeqShop:_Sequence_Mapping_and_Assembly_Practical#GotCloud Configuration File|SeqShop: Alignment: GotCloud Configuration File]] for more details
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See [[SeqShop:_Sequence Mapping and Assembly Practical, June 2014#GotCloud Configuration File|SeqShop: Alignment: GotCloud Configuration File]] for more details
 
* Note we want to limit snpcall to just chr22 so the configuration already has <code>CHRS = 22</code> (default was 1-22 & X).
 
* Note we want to limit snpcall to just chr22 so the configuration already has <code>CHRS = 22</code> (default was 1-22 & X).
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* Beagle will take about 2-3 minutes to complete
 
* Beagle will take about 2-3 minutes to complete
 
* Thunder will automatically run and will take another 3-4 minutes
 
* Thunder will automatically run and will take another 3-4 minutes
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<div class="mw-collapsible mw-collapsed" style="width:350px">
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When completed, it should look like this:
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<div class="mw-collapsible-content">
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[[File:GcldrefineOut.png]]
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</div>
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</div>
    
=== Genotype Refinement Output ===
 
=== Genotype Refinement Output ===
    
; What's new in the output directory?
 
; What's new in the output directory?
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<ul>
 
<ul>
 
<div class="mw-collapsible mw-collapsed" style="width:500px">
 
<div class="mw-collapsible mw-collapsed" style="width:500px">
 
<li>Answer</li>
 
<li>Answer</li>
 
<div class="mw-collapsible-content">
 
<div class="mw-collapsible-content">
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:<pre>ls ${OUT}</pre>
 
<ul>
 
<ul>
 
<li><code>beagle</code> directory : Beagle output</li>
 
<li><code>beagle</code> directory : Beagle output</li>
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</ul>
 
</ul>
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Let's take a look at that interesting location we found in the [[SeqShop:_Sequence_Mapping_and_Assembly_Practical#Accessing_BAMs_by_Position|alignment tutorial]] : chromosome 22, positions 36907000-36907100
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Let's take a look at that interesting location we found in the [[SeqShop:_Sequence_Mapping_and_Assembly_Practical, June 2014#Accessing_BAMs_by_Position|alignment tutorial]] : chromosome 22, positions 36907000-36907100
    
Use tabix to extract that from the VCFs:
 
Use tabix to extract that from the VCFs:
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Let's get some information on the BEAGLE VCF:
 
Let's get some information on the BEAGLE VCF:
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perl ${SS}/ext/bed-diff.pl --vcf1 ${SS}/ref22/1kg.omni.chr22.36Mb.vcf.gz --vcf2 ${OUT}/beagle/chr22/chr22.filtered.PASS.beagled.ALL.vcf.gz --gcRoot ${GC} --out ${OUT}/bedDiff.beagle
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perl ${EXT}/bed-diff.pl --vcf1 ${REF}/1kg.omni.chr22.36Mb.vcf.gz --vcf2 ${OUT}/beagle/chr22/chr22.filtered.PASS.beagled.ALL.vcf.gz --gcRoot ${GC} --out ${OUT}/bedDiff.beagle
      
Look at the results:
 
Look at the results:
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Now, let's see if it improved after running Thunder VCF:
 
Now, let's see if it improved after running Thunder VCF:
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  perl ${EXT}/bed-diff.pl --vcf1 ${REF}/1kg.omni.chr22.36Mb.vcf.gz --vcf2 ${OUT}/thunder/chr22/ALL/thunder/chr22.filtered.PASS.beagled.ALL.thunder.vcf.gz --gcRoot ${GC} --out ${OUT}/bedDiff.thunder
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  perl ${SS}/ext/bed-diff.pl --vcf1 ${SS}/ref22/1kg.omni.chr22.36Mb.vcf.gz --vcf2 ${OUT}/thunder/chr22/ALL/thunder/chr22.filtered.PASS.beagled.ALL.thunder.vcf.gz --gcRoot ${GC} --out ${OUT}/bedDiff.thunder
    
Look at the results:
 
Look at the results:
Retrieved from "http://genome.sph.umich.edu/wiki/Special:MobileDiff/11161...11991"

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