SeqShop: Calling Your Own Genome, December 2014

Login instructions for seqshop-server

Login to the seqshop-server Linux Machine

This section will appear redundantly in each session. If you are already logged in or know how to log in to the server, please skip this section

1. Login to the windows machine
• The username/password for the Windows machine should be written on the right-hand monitor
2. Start xming so you can open external windows on our Linux machine
• Start->Enter "Xming" in the search and select "Xming" from the program list
• Nothing will happen, but Xming was started.
View Screenshot



3. Open putty
• Start->Enter "putty" in the search and select "PuTTY" from the program list
View Screenshot



4. Configure PuTTY in the PuTTY Configuration window
• Host Name: seqshop-server.sph.umich.edu
View Screenshot



• Setup to allow you to open external windows:
• In the left pannel: Connection->SSH->X11
• Add a check mark in the box next to Enable X11 forwarding
View Screenshot



• Click Open
• If it prompts about a key, click OK
5. Enter your provided username & password as provided

You should now be logged into a terminal on the seqshop-server and be able to access the test files.

• If you need another terminal, repeat from step 3.

Login to the seqshop Machine

So you can each run multiple jobs at once, we will have you run on 4 different machines within our seqshop setup.

• You can only access these machines after logging onto seqshop-server

3 users logon to:

ssh -X seqshop1

3 users logon to:

ssh -X seqshop2

2 users logon to:

ssh -X seqshop3

2 users logon to:

ssh -X seqshop4

Tuesday - Start INDEL Calling

Setup

The indel & snpcall pipelines 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:

screen

As it says, press Space or Return.

• It should now look basically the same as your normal command line.

Set these values. Also, be sure to specify your sample name instead of SampleXX

export SAMPLE=SampleXX
source /net/seqshop-server/home/mktrost/seqshop/setupSS.txt

See the settings you just used:

cat /net/seqshop-server/home/mktrost/seqshop/setupSS.txt

Shows you:

export GC=/net/seqshop-server/home/mktrost/seqshop/gotcloud

export SS=/net/seqshop-server/home/mktrost/seqshop/singleSample

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

SampleXX SampleXX/output/bams/SampleXX.recal.bam

• 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 Indel

No special Configuration settings for Indel calling

cat ~/$SAMPLE/gotcloud.conf

You will see something like this:

# Cluster Settings
BATCH_TYPE = 
BATCH_OPTS = 

OUT_DIR = Sample13/output

# Align Settings
MAP_TYPE = BWA_MEM
BWA_THREADS = -t 24
FASTQ_LIST = fastq.list

# SNP Call Settings
UNIT_CHUNK = 20000000      # Chunk size of SNP calling : 20Mb
VCF_EXTRACT = /net/seqshop-server/home/mktrost/seqshop/singleSample/snpOnly.vcf.gz
MODEL_GLFSINGLE = TRUE
MODEL_SKIP_DISCOVER = FALSE
MODEL_AF_PRIOR = TRUE

EXT_DIR = /net/seqshop-server/home/mktrost/seqshop/singleSample/ext
EXT = $(EXT_DIR)/ALL.chrCHR.phase3.combined.sites.unfiltered.vcf.gz $(EXT_DIR)/chrCHR.filtered.sites.vcf.gz

Running Indel Calling

Run GotCloud indel with 6 jobs running in parallel

• Why 6?
  • You want to run as many as you can.
  • 5 of you on the machine - 5*6 = 30 jobs will be running in parallel on that machine

${GC}/gotcloud indel --conf $SAMPLE/gotcloud.conf --numjobs 6

• Only need the configuration & number of threads, 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

Wednesday

Checking if INDEL Completed

Logging Back in to Check Jobs

How do you log back into screen?

screen -r

This will resume an already running screen.

Verify you got a "completed successfully" message.

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

Need a BAM Index File?

Check your BAM files

ls $SAMPLE/output/bams/*bam

Check your BAI files

ls $SAMPLE/output/bams/*bai

Do you have the same number of BAM & BAI files?

• If so, then you are good to go.
• If you have more BAI's than BAM's - oops!!! (LET ME KNOW)
• If you have more BAM's than BAI's, we need to generate the index:

  $GC/bin/samtools index $SAMPLE/output/bams/YOUR_BAM_WITH_MISSING_BAI.bam

Detach From screen

Detach from screen. We will resume it again later when we start SNPCall.

Ctrl-a d

Structural Variation Tutorial

Now we are going to run the Structural Variation Practical

Please go to: SeqShop: Analysis of Structural Variation Practical, December 2014

We will Start SNP Calling after the practical.

Start SNP Calling

Resume screen

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.

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

SampleXX SampleXX/output/bams/SampleXX.recal.bam

• 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 SnpCall Configuration

cat ~/$SAMPLE/gotcloud.conf

You will see something like this:

# Cluster Settings
BATCH_TYPE = 
BATCH_OPTS = 

OUT_DIR = Sample13/output

# Align Settings
MAP_TYPE = BWA_MEM
BWA_THREADS = -t 24
FASTQ_LIST = fastq.list

# SNP Call Settings
UNIT_CHUNK = 20000000      # Chunk size of SNP calling : 20Mb
VCF_EXTRACT = /net/seqshop-server/home/mktrost/seqshop/singleSample/snpOnly.vcf.gz
MODEL_GLFSINGLE = TRUE
MODEL_SKIP_DISCOVER = FALSE
MODEL_AF_PRIOR = TRUE

EXT_DIR = /net/seqshop-server/home/mktrost/seqshop/singleSample/ext
EXT = $(EXT_DIR)/ALL.chrCHR.phase3.combined.sites.unfiltered.vcf.gz $(EXT_DIR)/chrCHR.filtered.sites.vcf.gz

Configuration Updates

No configuration updates from the original settings are necessary.

• Originally you were going to update the configuration to do exome calling only, but we have decided to do whole genome
  • If it doesn't finish tonight, we will kill it at the tutorial in the morning, and take advantage of the GotCloud restart capability after the tutorial tomorrow.

If you started making updates to your configuration yesterday, you can try to make it match above (mktrost is actually the path you want in the paths above).

• The only difference from above should be your OUT_DIR.

Check it with (leave as ~mktrost - you are comparing to my NA12878 conf file):

diff $SAMPLE/gotcloud.conf ~mktrost/NA12878/gotcloud.conf

If you see differences other than OUT_DIR, you can modify your file using nedit (or your favorite editor as you used yesterday).

• Or you can copy my file and just change OUT_DIR:

cp ~mktrost/NA12878/gotcloud.conf $SAMPLE/gotcloud.conf
nedit $SAMPLE/gotcloud.conf&

• Edit OUT_DIR

OUT_DIR = Sample##/output

• Save, and close

Running SnpCall

Run GotCloud snpcall with 6 jobs running in parallel

• Why 6?
  • You want to run as many as you can.
  • 5 of you on the machine - 5*6 = 30 jobs will be running in parallel on that machine

${GC}/gotcloud snpcall --conf $SAMPLE/gotcloud.conf --numjobs 6

• Only need the configuration & number of threads, 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

Thursday

Checking if SnpCall Completed

Logging Back in to Check Jobs

How do you log back into screen?

screen -r

This will resume an already running screen.

Checking Completion

Did you get a "completed successfully" message?

If yes, how long did SNP calling take? Look at the log message - time in seconds.

If no, are you running on seqshop-server? If so, KILL it. ssh -X to one of the seqshop machines and run on there.

Ctrl-c

Detach from screen. We will resume it again later when we restart SNPCall (if necessary).

Ctrl-a d

Ancestry Tutorial

Now we are going to run the Structural Variation Practical

Please go to: SeqShop: Estimates of Genetic Ancestry Practical, December 2014

We will Resume SNP Calling (if necessary) after the practical.

Restart SnpCall

Resume screen

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.

Running SnpCall

Run GotCloud snpcall with 6 jobs running in parallel

${GC}/gotcloud snpcall --conf $SAMPLE/gotcloud.conf --numjobs 6

• GotCloud will pick up after the last completed step from before.

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

Friday

SeqShop: Ancestry On Your Own Genome, December 2014

SeqShop: Ancestry On Your Own Genome, December 2014

Association Analysis Tutorial

Now we are going to run the Association Analysis Practical

Please go to: SeqShop: Genetic Association Analysis Practical, December 2014

We will look at our own genomes again after the practical.

Return to SeqShop: Ancestry On Your Own Genome, December 2014

Return to SeqShop:_Ancestry_On_Your_Own_Genome,_December_2014#Checking_if_Pileup_finished

Reviewing Indel Results

Set these values. Also, be sure to specify your sample name (or NA12878) instead of SampleXX

export SAMPLE=SampleXX
source /net/seqshop-server/home/mktrost/seqshop/setupSS.txt

Look in the output directory

ls ~/$SAMPLE/output

What in that directory was produced by indel calling?

• gotcloud.indel.conf
  • dump of all configuration settings for this run
• gotcloud.indel.Makefile
  • Makefile that was generated to manage all of the commands to be run
• gotcloud.indel.Makefile.log
  • log of all commands run by the Makefile
• indel/
  • 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.

Friday: Reviewing SNPCALL Results

Look in the output directory

ls ~/$SAMPLE/output

Look at the vcfs:

ls ~/$SAMPLE/output/vcfs

Friday : More SNP Analysis

Environmental Variables

If you didn't set the environmental variable, you can set it again

source /net/seqshop-server/home/mktrost/seqshop/setup.txt
export SAMPLE=SampleXX (MAKE SURE TO CHANGE XX to your number or use NA12878 instead)
source /net/seqshop-server/home/mktrost/seqshop/setupSS.txt

In addition, set another environmental variable for locating the binaries for custom analysis

export HK=/net/seqshop-server/home/hmkang/seqshop/bin

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.b138/dbsnp_138.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

$HK/run-command-wgs --cmd "$HK/vcf-add-rsid -vcf $OUT/vcfs/chr1/chr1.filtered.vcf.gz --db $HK/../data/dbSNP.b138/dbsnp_138.b37.vcf.gz --out $OUT/vcfs/chr1/chr1.filtered.rsid.vcf.gz" --numjobs 6

Looking up SNPs by rsID is possible by (for example)

$HK/vcf-lookup-rsid --vcf $OUT/vcfs/chr1/chr1.filtered.vcf.gz --sepchr --rs rs17766217

• 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 1,8,22 $HK/../data/gwascatalog/gwascatalog.txt | less

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

$HK/run-command-wgs --cmd "$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 

Extracting only exonic SNPs

If you want to look at the exonic SNPs, you can extract using the following command

$HK/run-command-wgs --cmd "($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

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

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 $GC/gotcloud.ref/human.g1k.v37.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