SeqShop: Variant Calling and Filtering for SNPs Practical, June 2014

Goals of This Session

• What we want to learn
  •  How to generate filtered variant calls for SNPs from BAMs
  • Basic variant call file format (VCF)
  •  How to examine the variants at particular genomic positions
  •  How to evaluate the quality of SNP calls

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

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

Open putty

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

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

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

Setup your run environment

This is the same setup you did for the previous tutorial, but you need to redo it each time you log in. It will setup some environment variables to point you to:

• GotCloud program
• Tutorial input files
• Setup an output directory
  • It will leave your output directory from the previous tutorial in tact.

source /home/mktrost/seqshop/setup.txt

• You won't see any output after running source
  • It silently sets up your environment

View setup.txt

Examining GotCloud SnpCall Input files

Reference Files

Reference files can be downloaded with GotCloud or from other sources

• See GotCloud: Genetic Reference and Resource Files for more information on downloading/generating reference files

For GotCloud snpcall, you need:

1. Reference genome FASTA file
   • Contains the reference base for each position of each chromosome
   • Additional information on the FASTA format: http://en.wikipedia.org/wiki/FASTA_format
2. VCF (variant call format) files with chromosomes/positions
   • dbsnp - used to skip known variants when recalibrating
   • hapmap - used for sample contamination/sample swap validation

GotCloud Configuration File

We will use the same configuration file as we used yesterday in GotCloud Align.

Run GotCloud SnpCall

Now that we have all of our input files, we need just a simple command to run:

${GC}/gotcloud/gotcloud snpcall --conf ${GC}/inputs/gotcloud.conf --numjobs 4 --region 22:36000000-37000000

• --numjobs tells GotCloud how many jobs to run in parallel
  • Depends on your system
• --region 22:36000000-37000000
  • The sample files are just a small region of chromosome 22, so to save time, we tell Gotcloud to ignore the other regions

This should take about 5 minutes to run.

It should end with a line like: TBD

If you cancelled GotCloud part way through, just rerun your GotCloud command and it will pick up where it left off.

Examining GotCloud SnpCall Output

Filtering Summary Statistics

cat ${OUTPUT}/vcfs/chr22/chr22.filtered.sites.vcf.summary

View Annotated Screenshot

GotCloud Genotype Refinement

seqshop/gotcloud/gotcloud beagle --conf seqshop/inputs/gotcloud.conf --numjobs 2 --region 22:36000000-37000000