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

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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

    Sequnce Alignment Files: BAM Files

    Reference Files

    Reference files can be downloaded with GotCloud or from other sources

    For GotCloud snpcall, you need:

    1. Reference genome FASTA file
    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.

    See SeqShop: Alignment: GotCloud Configuration File for more details

    Note we want to limit snpcall to just chr22 so the configuration already has CHRS = 22.

    GotCloud BAM Index File

    The BAM index file points GotCloud to the BAM files

    • Alignment pipeline generates for you
    • Tab delimited

    Look at the BAM index file the alignment pipeline generated 

    cat ${OUT}/bam.index
    What is the path to the BAM file for sample HG00640?
    • Answer:
      • /home/YourUserName/out/bams/HG00640.recal.bam
        •  

    The alignment pipeline only processed 4 samples, but for snpcall, we want to run on 62 samples.

    • The other 58 samples were already aligned:
    ls ${IN}/bams

    Look at the BAM index for those BAMs: 

    less ${IN}/bams/bam.index

    Remember, use 'q' to exit out of less 

    q
    Do you notice a difference between this index and yours?
    • Answer:
      • It doesn't have a full path to the BAM file, while your index has /home/...
        •  
      • That's ok, gotcloud.conf contains the path to those BAMs
        •  


    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
    cat ${IN}/bams/bam.index >> ${OUT}/bam.index
    • ">>" will append to the file that follows it

    Verify your BAM index contains the additional BAMs 

     less ${IN}/bams/bam.index

    Remember, use 'q' to exit out of less 

    q
    Do you see both sets of BAMs?
    • Screenshot:
      • If not, let me know
        •  

    Run GotCloud SnpCall

     

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

    ${GC}/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
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