View source for SeqShop: Calling Your Own Genome, May 2015

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''Login instructions for seqshop-server''
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{{SeqShopLogin}}
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== Tuesday - Start SNP Calling ==
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=== Setup Screen ===
The snpcall pipeline will run overnight, but you'll want to log out.
; How do I leave something running on the server even if I log out?
: One solution is screen!

; How do I use screen?
: Before running your command, you need to start screen:
: <pre>screen</pre>

[[File:Screen.png]]

As it says, press <code>Space</code> or <code>Return</code>.
* It should now look basically the same as your normal command line.

=== Setup Variables ===
Set these values.  Also, be sure to specify your sample name instead of SampleXX
 export SAMPLE=SampleXX
or
 export SAMPLE=NA12878

Point to your GotCloud & your output directory:
 export GC=~/seqshop/gotcloud
 export OUT=~/$SAMPLE/output

=== List of BAMs ===
The list of BAMs has already been created (just 1 BAM, your sample).
* But it is simply SAMPLE\tBAM_name, so easy to figure out

 cat ~/$SAMPLE/output/bam.list

:<code>SampleXX   SampleXX/output/bams/SampleXX.recal.bam</code>
* Relative path, so assumes running from your home directory (I prefer absolute paths, but for simplicity of the workshop, we just use relative path).

=== Configuring SNPCALL ===

 cat ~/$SAMPLE/gotcloud.conf

You will see this:
<pre>
# References
SS_DIR = /net/seqshop-server/home/mktrost/seqshop/singleSample
REF_DIR = $(SS_DIR)/ref/gotcloud.ref/

######### ALIGNMENT ########
MAP_TYPE = BWA_MEM
FASTQ_LIST = fastq.list
BATCH_TYPE = 
BATCH_OPTS = 
BWA_THREADS = -t 6

# SNP Call Settings
UNIT_CHUNK = 20000000      # Chunk size of SNP calling : 20Mb
VCF_EXTRACT = $(SS_DIR)/snpOnly.vcf.gz
MODEL_GLFSINGLE = TRUE
MODEL_SKIP_DISCOVER = FALSE
MODEL_AF_PRIOR = TRUE

EXT_DIR = $(SS_DIR)/ext
EXT = $(EXT_DIR)/ALL.chrCHR.phase3.combined.sites.unfiltered.vcf.gz $(EXT_DIR)/chrCHR.filtered.sites.vcf.gz
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=== Running SNP Calling ===
Run GotCloud snpcall with 8 jobs running in parallel
* Why 8?  
** You want to run as many as you can.
** 2-3 of you on the machine - 3*8 = 24 jobs will be running in parallel on that machine
 ${GC}/gotcloud snpcall --conf $SAMPLE/gotcloud.conf --numjobs 8 --outdir $OUT
* Only need the configuration, number of threads, and the output directory, rest is specified within the configuration.

This will run overnight.  We will check if it completed at the practical in the morning.

=== Log Out ===
;Want to log out and leave your job running?
In the screen window, type:
 Ctrl-a d
(Hold down Ctrl and type 'a', let go of both and type 'd')
* This will "detach" from your screen session while your alignment continues to run.

If you have not detached from screen:
 Ctrl-a d

exit PuTTY


=== FEEDBACK! ===
Please provide feedback on today:

https://docs.google.com/forms/d/1ADTkBjzT-QNj2lrejyqGqDaahTponrw20kSgDNwqwH4/viewform

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== Thursday ==
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=== Checking if snpcall Completed ===
==== Resume screen to Check Jobs ====

;How do you log back into screen?
 screen -r
This will resume an already running screen.

Your screen session still has your environment variables set, so you do not need to reset them.


Verify you got a "completed successfully" message.

How long did snpcall calling take?  Look at the log message - time in seconds.


==== List of BAMs ====
The list of BAMs has already been created (just 1 BAM, your sample).
* But it is simply SAMPLE\tBAM_name, so easy to figure out

 cat ~/$SAMPLE/output/bam.list

:<code>SampleXX   SampleXX/output/bams/SampleXX.recal.bam</code>
* Relative path, so assumes running from your home directory (I prefer absolute paths, but for simplicity of the workshop, we just use relative path).


==== GotCloud INDEL Configuration ====

 cat ~/$SAMPLE/gotcloud.conf

Same as it looked the other day with no special Configuration settings for INDEL calling.

==== Running INDEL ====
Run GotCloud indel with 6 jobs running in parallel
 ${GC}/gotcloud indel --conf $SAMPLE/gotcloud.conf --numjobs 6 --outdir $OUT
* Only need the configuration, number of threads, and the output directory, rest is specified within the configuration.

This will run overnight.  We will check if it completed at the practical in the morning.

==== Log Out ====
;Want to log out and leave your job running?
In the screen window, type:
 Ctrl-a d
(Hold down Ctrl and type 'a', let go of both and type 'd')
* This will "detach" from your screen session while your alignment continues to run.

exit PuTTY

=== FEEDBACK!===
Please provide feedback for today.
https://docs.google.com/a/umich.edu/forms/d/1iES6usHxLB7Ec9hRxtqYgH7v05lU3Ume4VJcksx8Ogg/viewform

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== Friday ==
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=== SeqShop: Ancestry On Your Own Genome, May 2015 ===
[[SeqShop: Ancestry On Your Own Genome, May 2015]]

=== Setup Variables ===
Set these values.  Also, be sure to specify your sample name instead of SampleXX
 export SAMPLE=SampleXX
or
 export SAMPLE=NA12878

Point to your GotCloud & your output directory:
 export GC=~/seqshop/gotcloud
 export OUT=~/$SAMPLE/output

=== Reviewing Indel Results ===
Look in the output directory
 ls ~/$SAMPLE/output

What in that directory was produced by indel calling?
* <code>gotcloud.indel.conf</code>
** dump of all configuration settings for this run
* <code>gotcloud.indel.Makefile</code>
** Makefile that was generated to manage all of the commands to be run
* <code>gotcloud.indel.Makefile.log</code>
** log of all commands run by the Makefile
* <code>indel/</code>
** indel output directory

Let's look at the indel output
 ls ~/$SAMPLE/output/indel 
* 3 directories
** aux - intermediate files
** indelvcf - intermediate files
** '''final indel files'''

Final indel directory:
 ls ~/$SAMPLE/output/indel/final
* '''all.genotypes.vcf.gz - output VCF'''
* '''all.genotypes.vcf.gz.tbi - output VCF index file to allow jumping to positions'''
* all.genotypes.vcf.gz.tbi.OK - completion indicator
* merge/ - directory with per chromosome bcf (binary vcf) files
* all.genotypes.vcf.gz.OK - completion indicator
* all.genotypes.vcf.gz.tbi.log - log
* concat.log - log

==== Looking at final INDEL VCF ====

Note that because this is a single sample calling, many of the INFO fields are less meaningful as many of the values like HWE p values, allele frequencies, inbreeding coefficient are a function of a population.
Nonetheless, we may examine the results.  First, we see how many indels were discovered for your genome:

 $GC/bin/vt peek ~/$SAMPLE/output/indel/final/all.genotypes.vcf.gz 

       no. Indels                         :     588566
           2 alleles (ins/del)            :          588566 (0.87) [273261/315305]

This gives use 588,566 indels with an insertion deletion ratio of 0.87.

We next look at the filtered set. The PASS filter reduces the setof indels to a non overlapping set and the INFO.AC!=0 extracts all indels that are either heterozygous or homozygous alternative.
Some indels that were originally discovered were found to be the homozygous reference genotype.  Invariably, these are relative high depth calls where the 
alternative allele is discovered less or is mis-specified.

 $GC/bin/vt peek ~/$SAMPLE/output/indel/final/all.genotypes.vcf.gz -f "PASS&&INFO.AC!=0"

       no. Indels                         :     549963
           2 alleles (ins/del)            :          549963 (0.91) [261480/288483]

About 38K indels were removed, the insertion deletion ratio increases to 0.91.  Note that in general, for high depth data, discovered indels are reported with insertion deletion ratios
close to 1. So this is a good sign.  Next generation sequencing errors are bias for deletions.

It is possible to perform a slightly more stringent filtering using allele balance.  The allele balance estimator in this case is meaningful still for an individual because it is a function of read depth.
Note that AB>0.5 denotes reference bias and AB<0.5 denotes alternative allele bias.

 $GC/bin/vt peek ~/$SAMPLE/output/indel/final/all.genotypes.vcf.gz -f "PASS&&INFO.AC>0&&INFO.AB<0.7&&INFO.AB>0.3"

       no. Indels                         :     490965
           2 alleles (ins/del)            :          490965 (0.92) [235254/255711]

The insertion deletion ratio increases from 0.91 to 0.92.  

=== Return to SeqShop: Ancestry On Your Own Genome, May 2015 ===
Return to [[SeqShop:_Ancestry_On_Your_Own_Genome,_May_2015#Checking_if_Pileup_finished]]

=== Friday: Reviewing SNPCALL Results ===
Look in the output directory
 ls ~/$SAMPLE/output

Look at the vcfs:
 ls ~/$SAMPLE/output/vcfs

=== Friday : More SNP Analysis ===
In addition, set another environmental variable for locating the binaries for custom analysis

 export HK=/net/seqshop-server/home/hmkang/apigenome/bin
 export EPACTS=/net/seqshop-server/home/mktrost/seqshop/epacts/
 export REF=/net/seqshop-server/home/mktrost/seqshop/singleSample/ref/gotcloud.ref
 export GC=~/seqshop/gotcloud


 export SAMPLE=SampleXX 
 export OUT=~/$SAMPLE/output

==== Annotation / Lookup against dbSNP ====

If you want to add rsIDs to your variant files, you can do this by running the following command

 $HK/vcf-add-rsid -vcf $OUT/vcfs/chr1/chr1.filtered.vcf.gz --db $HK/../data/dbsnp_142.b37.vcf.gz --out $OUT/vcfs/chr1/chr1.filtered.rsid.vcf.gz
 
If you want to run this command across all chromosomes in parallel, you can use the special script run-command-wgs

 $HK/run-make --repeat-chr --cmd "$HK/vcf-add-rsid -vcf $OUT/vcfs/chr1/chr1.filtered.vcf.gz --db $HK/../data/dbsnp_142.b37.vcf.gz --out $OUT/vcfs/chr1/chr1.filtered.rsid.vcf.gz" --numjobs 6 --out runmake.rsid

Looking up SNPs by rsID is possible by (for example, rs17766217) -- How can we find its position? 
 $HK/tabix ~/NA12878/output/vcfs/chr8/chr8.filtered.rsid.vcf.gz 8:128504497 | less
* Be sure to look at the QUAL & your sample's PL, and not just the GL field.  Check if QUAL is 0 or PL is 0,0,0 - NS is also probably 0; DP is probably 0.  That means you probably didn't have any copies, so your GT may not be correct/is unknown.

If you want to browse the rsIDs of known GWAS SNPs, you can do this by
 cut -f 1,8,12,13,22 $HK/../data/gwascatalog/gwascatalog.txt | grep -w rs17766217

==== Annotating your genome ====

You can annotate your genome using EPACTS software packages. Individual chromosome can be annotated by running.
 $EPACTS/bin/epacts anno --in $OUT/vcfs/chr1/chr1.filtered.rsid.vcf.gz --out $OUT/vcfs/chr1/chr1.filtered.rsid.anno.vcf.gz

Or you can run multiple chromosomes in parallel in one command
 $HK/run-make --repeat-chr --cmd "$EPACTS/bin/epacts anno --in $OUT/vcfs/chr1/chr1.filtered.rsid.vcf.gz --out $OUT/vcfs/chr1/chr1.filtered.rsid.anno.vcf.gz" --numjobs 6 --out runmake.anno

==== Extracting only exonic SNPs ====

If you want to look at the exonic SNPs, you can extract using the following command
 $HK/run-make --repeat-chr --cmd "($HK/tabix -H $OUT/vcfs/chr1/chr1.filtered.rsid.anno.vcf.gz; zcat $OUT/vcfs/chr1/chr1.filtered.rsid.anno.vcf.gz | grep Exon;)| $HK/bgzip -c > $OUT/vcfs/chr1/chr1.filtered.rsid.anno.exon.vcf.gz" --numjobs 6 --out runmake.exome

And they can be combined as follows
 (zcat $OUT/vcfs/chr1/chr1.filtered.rsid.anno.exon.vcf.gz; zcat $OUT/vcfs/chr[2-9]/chr*.filtered.rsid.anno.exon.vcf.gz $OUT/vcfs/chr??/chr*.filtered.rsid.anno.exon.vcf.gz $OUT/vcfs/chrX/chrX.filtered.rsid.anno.exon.vcf.gz | grep -v ^#) | $HK/bgzip -c > $OUT/wgs.filtered.rsid.anno.exon.vcf.gz
 $HK/tabix -pvcf $OUT/wgs.filtered.rsid.anno.exon.vcf.gz

==== Exonic Variants NOT found by 1000G ====

If you are interested in rare variants that are not identified by 1000G, you can extract them by running
 zcat $OUT/wgs.filtered.rsid.anno.exon.vcf.gz | grep "EXTFILTER=NA,NA" | grep -v -w "0/0" | less
 
For example, 
 zcat $OUT/wgs.filtered.rsid.anno.exon.vcf.gz | grep "EXTFILTER=NA,NA" | grep -v -w "0/0" | perl -lane 'print "$1\t$F[6]" if ( /ANNO=([^;:]+)/)' | sort | uniq -c
 
will give you the counts of variants, separate by the filtering results

* Q1. How manny novel silent, missense, and nonsense SNPs are found? Is that too few, too small, or just about right?
* Q2. Looking at each functional category, which functional categories has largest fraction of SNPs failed filter? Why do you think it is?
* Q3. Can you exclude the sites that are also in dbSNP, and count how many nonsense variants are left?


To also exclude those in dbsnp:
 zcat $OUT/wgs.filtered.rsid.anno.exon.vcf.gz | grep "EXTFILTER=NA,NA" | grep -v -w "0/0" | grep -v rs| perl -lane 'print "$1\t$F[6]" if ( /ANNO=([^;:]+)/)' | sort | uniq -c

Exclude dbsnp and look at Stop_Gain variants
 zcat $OUT/wgs.filtered.rsid.anno.exon.vcf.gz | grep "EXTFILTER=NA,NA" | grep -v -w "0/0" |grep -v rs | perl -lane 'print "$_" if ( /ANNO=Stop_Gain/)' |grep -w PASS

Want to see this from the BAM file?  Use samtools tview:
 $GC/bin/samtools tview $SAMPLE/output/bams/$SAMPLE.recal.bam $REF/hs37d5.fa
Use 'g' & enter the Chr:Pos
* Some patterns may indicate not real variants.

If you want to know predicted functional significance of a particular variant, you can search by

 $HK/tabix $HK/../data/CADD/whole_genome_SNVs.tsv.gz [chr]:[pos] | head -3
 
The phred score at the last column quantifies the degree of functional significance


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== OVERALL COURSE FEEDBACK! ==
Please provide feedback:
https://docs.google.com/forms/d/1pxfPXKwWfA71ZJM99Sevs3MwAUz2UbHAR8dnRI-kRNM/viewform