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SeqShop: Genetic Association Analysis Practical, December 2014

Contents

Introduction

Goals of This Session

  • Understand how to annotate variants using EPACTS
  • Understand how to run single variant association analysis using EPACTS
  • Understand how to run rare variant association test using EPACTS
  • Understand how to visualize the association output from EPACTS

Setup in person at the SeqShop Workshop

This section is specifically for the SeqShop Workshop computers.

If you are not running during the SeqShop Workshop, please skip this section.

If you are not already logged in, please expand this section.

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
    • Xming.png

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

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

    • 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
        • SeqshopX11.png

    • 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.

    This 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 /net/seqshop-server/home/mktrost/seqshop/setup.txt
    
    • You won't see any output after running source
      • It silently sets up your environment
      • If you want to view the detail of the setup, type
    less /net/seqshop-server/home/mktrost/seqshop/setup.txt
    

    and press 'q' to finish.

    View setup.txt

    export GC=/net/seqshop-server/home/mktrost/seqshop/gotcloud
    export SS=/net/seqshop-server/home/mktrost/seqshop/example
    export EPACTS=/net/seqshop-server/home/mktrost/seqshop/epacts
    export OUT=~/out
    mkdir -p ${OUT}
    

    Setup when running on your own outside of the SeqShop Workshop

    This section is specifically for running on your own outside of the SeqShop Workshop.

    If you are running during the SeqShop Workshop, please skip this section.

    This tutorial builds on the alignment & snpcall tutorials, if you have not already, please first run those tutorials: Alignment Tutorial & SNP Calling Tutorial

    Download & Build EPACTS

    If you do not already have EPACTS:

    • cd to where you want EPACTS installed (you can change this to any directory you want)
    mkdir -p ~/seqshop
    cd ~/seqshop/
    
    • download, decompress, and build the version of epacts that was tested with this tutorial:
    wget http://csg.sph.umich.edu//kang/epacts/download/EPACTS-3.2.6.tar.gz
    tar xvf EPACTS-3.2.6.tar.gz
    cd EPACTS-3.2.6
    ./configure --prefix=$HOME/seqshop/epacts
    make
    make install
    cd ../..
    

    Setup your run environment

    Environment variables will be used throughout the tutorial.

    We recommend that you setup these variables so you won't have to modify every command in the tutorial.

    1. Point to where you installed GotCloud
    2. Point to where you installed the seqshop files
    3. Point to where you want the output to go
    Using bash (replace the paths below with the appropriate paths):
    export GC=~/seqshop/gotcloud
    export SS=~/seqshop/example
    export OUT=~/seqshop/output
    Using tcsh (replace the paths below with the appropriate paths):
    setenv GC ~/seqshop/gotcloud
    setenv SS ~/seqshop/example
    setenv OUT ~/seqshop/output
    • Additional variables for EPACTS:
      • Using bash (replace the paths below with the appropriate paths):
      • export EPACTS=~/seqshop/epacts
      • Using tcsh (replace the paths below with the appropriate paths):
      • setenv EPACTS ~/seqshop/epacts

    Preparing Input Files

    Input VCF file

    We will use SNP genotypes from the SNP calling session, after LD-aware genotype refinement. Check the contents of the VCF file using the following command.

    zless ${OUT}/thunder/chr22/ALL/thunder/chr22.filtered.PASS.beagled.ALL.thunder.vcf.gz
    

    Phenotype Information

    Phenotype information is prepared in PED format commonly used in other GWAS software such as MERLIN or PLINK.

    less ${SS}/assoc/seqshop.ped
    

    The first several line should look like below.

    View Data

    #FAM_ID	IND_ID	DAD_ID	MOM_ID	SEX	PHENO
    HG00551	HG00551	0	0	0	0
    HG00553	HG00553	0	0	0	0
    HG00554	HG00554	0	0	0	0
    HG00637	HG00637	0	0	0	0
    HG00638	HG00638	0	0	0	0
    HG00640	HG00640	0	0	0	1
    HG00641	HG00641	0	0	0	1
    HG00734	HG00734	0	0	0	1
    HG00736	HG00736	0	0	0	0
    ...
    

    Binary phenotype can be encoded as 0-1 or 1-2. If the column contains more than two distinct values, it will automatically be recognized as quantitative values.

    EPACTS allows PED file to have a header line. The header line should contain the description of each column. EPACTS also accepts a standard PED format where .ped file contains the phenotype data and .dat file contains the information about each column.

    Installed version of EPACTS

    EPACTS are installed in the server. If you want to install EPACTS by yourself, visit EPACTS page for more details

    ls $EPACTS/bin
    

    View EPACTS executable files

    anno   epacts       epacts-cis-extract  epacts-group       epacts-multi     epacts.pm      epstopdf  test_run_epacts.sh
    bgzip  epacts-anno  epacts-download     epacts-make-group  epacts-pca-plot  epacts-single  pEmmax    vcfast
    chaps  epacts-cat   epacts-enrich       epacts-make-kin    epacts-plot      epacts-zoom    tabix     wGetOptions.pm
    

    Note that some tools undocumented in EPACTS documentation is under development and may not work.

    Annotating Variants with EPACTS

    There are multiple software tools that provides a function to annotate variants, such as Variant Effect Predictor (VEP) that is used in 1000 Genomes Project. While most annotation software provides very similar results to each other, their computational efficiency can substantially vary. The annotation software EPACTS provides is extremely fast and can provide genome-wide annotation results in orders of magnitude faster than other widely available annotation software.

    In order to annotate variants with EPACTS, one can use epacts-anno module.

    mkdir --p $OUT/assoc
    $EPACTS/bin/epacts-anno --in $OUT/thunder/chr22/ALL/thunder/chr22.filtered.PASS.beagled.ALL.thunder.vcf.gz --out $OUT/assoc/snps.anno.vcf.gz --ref $SS/ref22/human.g1k.v37.chr22.fa
    

    Then you will see a series of messages before annotation finishes.

    View the expected messages

    /home/hmkang/seqshop/epacts/bin/anno -i /net/seqshop-server/hmkang/out/thunder/chr22/ALL/thunder/chr22.filtered.PASS.beagled.ALL.thunder.vcf.gz -r \
       /home/hmkang/seqshop/ref22/human_g1k_v37.chr22.fa -f refGene -g /home/hmkang/seqshop/epacts/share/EPACTS/hg19_gencodeV14.txt.gz \
      -c /home/hmkang/seqshop/epacts/share/EPACTS/codon.txt -o  /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz --inputFormat vcf \
      -p /home/hmkang/seqshop/epacts/share/EPACTS/priority.txt
    The following parameters are available.  Ones with "[]" are in effect:
    
    Available Options
        Required Parameters :
                             -i [/net/seqshop-server/hmkang/out/thunder/chr22/ALL/thunder/chr22.filtered.PASS.beagled.ALL.thunder.vcf.gz]
                             -o [/net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz]
             Gene Annotation
                Parameters : -g [/home/hmkang/seqshop/epacts/share/EPACTS/hg19_gencodeV14.txt.gz]
                             -r [/home/hmkang/seqshop/ref22/human_g1k_v37.chr22.fa]
                             --inputFormat [vcf], --checkReference, -f [refGene]
                             -p [/home/hmkang/seqshop/epacts/share/EPACTS/priority.txt]
                             -c [/home/hmkang/seqshop/epacts/share/EPACTS/codon.txt]
                             -u [], -d [], --se [], --si [], --outputFormat []
     Other Annotation Tools : --genomeScore [], --bed [], --tabix []
    Load reference genome /home/hmkang/seqshop/ref22/human_g1k_v37.chr22.fa...
    DONE: 1 chromosomes and 51304566 bases are loaded.
    Load codon file /home/hmkang/seqshop/epacts/share/EPACTS/codon.txt...
    DONE: codon file loaded.
    Load priority file /home/hmkang/seqshop/epacts/share/EPACTS/priority.txt...
    DONE: 24 priority annotation types loaded.
    Load gene file /home/hmkang/seqshop/epacts/share/EPACTS/hg19_gencodeV14.txt.gz...
    DONE: 92627 gene loaded.
    DONE: Generated frequency of each annotype type in [ /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz.anno.frq ].
    DONE: Generated frequency of each highest priority annotation type in [ /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz.top.anno.frq ].
    Ts/Tv ratio: 2.35733
    Ts observed: 2718  times; Tv observed: 1153 times.
    DONE: Generated frequency of each base change in [ /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz.base.frq ].
    DONE: Generated frequency of each codon change in [ /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz.codon.frq ].
    DONE: Generated frequency of indel length in [ /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz.indel.frq ].
    ..............................................         
     ...      Anno(tation)                       ...       
     ...      Xiaowei Zhan, Goncalo Abecasis     ...      
      ...      Speical Thanks:                    ...     
       ...      Hyun Ming Kang, Yanming Li         ...    
        ...      zhanxw@umich.edu                    ...  
         ...      Sep 2011                            ... 
          ................................................
                                                          
    DONE: 3871 varaints are annotated.
    DONE: Generated annotation output in [ /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz ].
    Annotation succeed!
    mv /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz /net/seqshop-server/hmkang/out/thunder/chr22/ALL/thunder/chr22.filtered.PASS.beagled.ALL.thunder.vcf.gz.tmp
    /home/hmkang/seqshop/epacts/bin/bgzip -c /net/seqshop-server/hmkang/out/thunder/chr22/ALL/thunder/chr22.filtered.PASS.beagled.ALL.thunder.vcf.gz.tmp > /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz
    /home/hmkang/seqshop/epacts/bin/tabix -pvcf -f /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz
    rm /net/seqshop-server/hmkang/out/thunder/chr22/ALL/thunder/chr22.filtered.PASS.beagled.ALL.thunder.vcf.gz.tmp
    rm /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz.log /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz.top.anno.frq /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz.anno.frq /net/seqshop- server/hmkang/out/assoc/snps.anno.vcf.gz.base.frq /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz.codon.frq /net/seqshop-server/hmkang/out/assoc/snps.anno.vcf.gz.indel.frq
    

    After running annotation, you can check the annotation results. Let's look at the APOL g1 risk allele we manually examined in the SNP calling section.

    $GC/bin/tabix $OUT/assoc/snps.anno.vcf.gz 22:36661906 | head -1 | cut -f 1-8
    

    View the annotation results

    22	36661906	.	A	G	18	PASS	DP=409;MQ=59;NS=62;AN=124;AC=2;AF=0.013827;AB=0.4065;AZ=-0.5287;FIC=-0.0092;
                SLRT=-0.0075;HWEAF=0.0138;HWDAF=0.0276,0.0000;LBS=36,36,0,0,1,1,0,0;OBS=145,191,0,0,3,2,0,0;STR=-0.040;
                STZ=-0.740;CBR=0.008;CBZ=0.144;IOR=0.000;IOZ=-1.370;AOI=-5.614;AOZ=-4.243;LQR=0.178;MQ0=0.000;MQ10=0.000;MQ20=0.000;
                MQ30=0.000;SVM=1.51214;BAVGPOST=0.998;BRSQ=0.941;LDAF=0.0161;AVGPOST=1.0000;RSQ=1.0000;ERATE=0.0019;THETA=0.0013;
               ANNO=Nonsynonymous:APOL1;ANNOFULL=APOL1/ENST00000397278.3:+:Nonsynonymous(AGC/Ser/S->GGC/Gly/G:Base1025/1197:Codon342/399:Exon6/6):Exon|
               APOL1/ENST00000426053.1:+:Nonsynonymous(AGC/Ser/S->GGC/Gly/G:Base971/1143:Codon324/381:Exon5/5):Exon|
               APOL1/ENST00000422706.1:+:Nonsynonymous(AGC/Ser/S->GGC/Gly/G:Base1025/1197:Codon342/399:Exon6/6):Exon|
               APOL1/ENST00000319136.4:+:Nonsynonymous(AGC/Ser/S->GGC/Gly/G:Base1073/1245:Codon358/415:Exon7/7):Exon|
               APOL1/ENST00000347595.7:+:Nonsynonymous(AGC/Ser/S->GGC/Gly/G:Base662/834:Codon221/278:Exon3/3):Exon|
               APOL1/ENST00000397279.4:+:Nonsynonymous(AGC/Ser/S->GGC/Gly/G:Base1025/1197:Codon342/399:Exon6/7):Exon
    
    • What is the function of this variant?
    • How many different transcript does the variant overlap with?
    • How can you represent the variant in terms of amino acid changes?

    Single Variant Association Analysis

    Let's run a single-variant association analysis using a score test.

    $EPACTS/bin/epacts single --ped $SS/assoc/seqshop.ped --vcf $OUT/assoc/snps.anno.vcf.gz --out $OUT/assoc/single --region 22:36000000-37000000 --test b.score --min-mac 1 --pheno PHENO --run 2
    

    After running it, you will see EPACTS output files by looking at

    ls $OUT/assoc
    

    The top association results can be viewed by

    head $OUT/assoc/single.epacts.top5000
    

    View top association results

    #CHROM	BEGIN	END	MARKER_ID	NS	AC	CALLRATE	MAF	PVALUE	SCORE	NS.CASE	NS.CTRL	AF.CASE	AF.CTRL
    22	36995620	36995620	22:36995620_A/G	62	36	1	0.29032	5.6717e-09	5.8262	31	31	0.51613	0.064516
    22	36993088	36993088	22:36993088_G/C	62	30	1	0.24194	7.3258e-07	4.9525	31	31	0.43548	0.048387
    22	36997871	36997871	22:36997871_G/T	62	30	1	0.24194	7.3258e-07	4.9525	31	31	0.43548	0.048387
    22	36987368	36987368	22:36987368_G/A	62	31	1	0.25	2.0898e-06	4.7445	31	31	0.43548	0.064516
    22	36987861	36987861	22:36987861_A/G	62	31	1	0.25	2.0898e-06	4.7445	31	31	0.43548	0.064516
    22	36985499	36985499	22:36985499_C/T	62	29	1	0.23387	5.7389e-06	4.5358	31	31	0.40323	0.064516
    22	36998907	36998907	22:36998907_C/T	62	58	1	0.46774	1.3679e-05	-4.3489	31	31	0.25806	0.67742
    22	36978260	36978260	22:36978260_G/T	62	28	1	0.22581	1.5051e-05	4.3279	31	31	0.3871	0.064516
    22	36667082	36667082	22:36667082_T/G	62	27	1	0.21774	0.00015573	-3.7817	31	31	0.064516	0.37097
    

    Interpretation for top associated variant chr22:36995620_A/G:

    #CHROM	BEGIN	END	MARKER_ID	NS	AC	CALLRATE	MAF	PVALUE	SCORE	NS.CASE	NS.CTRL	AF.CASE	AF.CTRL
    22	36995620	36995620	22:36995620_A/G	62	36	1	0.29032	5.6717e-09	5.8262	31	31	0.51613	0.064516
    
    The score test PVALUE = 5.6717e-09 is strongly significant.
    For the notation chr22:36995620_A/G, the reference allele is the first allele "A", and the non-reference (effect) allele is the second allele "G".
    The SCORE statistic = 0.29032 is positive (e.g. SCORE>0), meaning that the effect allele INCREASES your risk for disease.  Note that the SCORE is NOT the BETA or log odds ratio.  To calculate the BETA and SE(BETA), you must run the b.wald test.
    There is a large difference between the allele frequencies for cases and controls.  AF.CASE = 0.51613 >> AF.CTRL = 0.0064516.
    

    You can look also visualize the results by QQ-plot and Manhattan plot

    View QQ plots

    evince $OUT/assoc/single.epacts.qq.pdf&

    Single.epacts.qq.png

    View Manhattan plots

    evince $OUT/assoc/single.epacts.mh.pdf&

    Single.epacts.mh.png

    Also, you can create a zoom plot focusing on the region of interest

    $EPACTS/bin/epacts-zoom --vcf $OUT/assoc/snps.anno.vcf.gz --pos 22:36995620 --prefix $OUT/assoc/single
    

    View Zoom Plots

    evince $OUT/assoc/single.zoom.22.36995620.pdf&

    Single.zoom.22.36995620.png

    If you want to run EMMAX, you first need to create a kinship matrix

    $EPACTS/bin/epacts-make-kin --vcf $OUT/assoc/snps.anno.vcf.gz --min-maf 0.01 --out $OUT/assoc/snps.anno.kinf --run 2 --chr 22
    

    And run EMMAX test specifying the kinship matrix

    $EPACTS/bin/epacts-single --ped $SS/assoc/seqshop.ped --vcf $OUT/assoc/snps.anno.vcf.gz --out $OUT/assoc/emmax --region 22:36000000-37000000 --test q.emmax --pheno PHENO --run 2 --kinf $OUT/assoc/snps.anno.kinf
    

    Then the results may look similar to previous ones.

    head $OUT/assoc/emmax.epacts.top5000
    

    Run Groupwise Test

    To run group-wise test such as gene-level burden test, you need to create a marker group file. If your VCF is already annotated, you can create a group file by running

    $EPACTS/bin/epacts make-group --vcf $OUT/assoc/snps.anno.vcf.gz --out $OUT/assoc/snps.anno.grp --nonsyn
    

    The group file is simply a list of marker per group name, as shown below.

    cat $OUT/assoc/snps.anno.grp
    
    APOL1	22:36655735_G/A	22:36657740_G/A	22:36661330_G/A	22:36661566_G/A	22:36661646_G/A	22:36661891_G/A	22:36661906_A/G
    APOL2	22:36623731_T/C	22:36623920_G/A	22:36629466_T/A	22:36633107_C/A
    APOL3	22:36537763_C/T	22:36537798_G/A	22:36556768_G/A	22:36556823_G/T
    APOL4	22:36587154_G/T	22:36587202_G/A	22:36587223_G/T	22:36587346_C/T	22:36587511_C/T	22:36587704_T/C	22:36587886_C/T	22:36593714_G/A	22:36597744_A/C	22:36598049_C/G	22:36598058_T/C	22:36598081_A/T
    APOL5	22:36122356_G/A	22:36122380_T/A	22:36122930_C/T	22:36123083_C/T	22:36124860_C/G
    FOXRED2	22:36900271_T/C	22:36900806_A/G
    MYH9	22:36681163_G/C	22:36684354_T/C	22:36710183_T/C
    Metazoa_SRP	22:36711990_C/G
    RBFOX2	22:36424450_A/C
    RP4-633O19__A.1	22:36792162_G/A
    

    If you have your own annotation, you can create your own burden test unit by modifying this file.

    If you want to run a collapsing burden test (CMC), run the following command

    $EPACTS/bin/epacts group --ped $SS/assoc/seqshop.ped --vcf $OUT/assoc/snps.anno.vcf.gz --out $OUT/assoc/group.collapse --test b.collapse --groupf $OUT/assoc/snps.anno.grp --pheno PHENO --run 2 
    

    You can view the results by examining the output file

    cat $OUT/assoc/group.collapse.epacts
    

    View Output file

    #CHROM  BEGIN   END     MARKER_ID       NS      FRAC_WITH_RARE  NUM_ALL_VARS    NUM_PASS_VARS   NUM_SING_VARS   PVALUE  BETA    SEBETA  ZSTAT
    22      36655735        36661906        22:36655735-36661906_APOL1      62      0.14516 7       4       0       0.28783 0.80648 0.75876 1.0629
    22      36623731        36633107        22:36623731-36633107_APOL2      62      0.064516        4       1       0       0.99286 -17.704 1978.1  -0.0089502
    22      36537763        36556823        22:36537763-36556823_APOL3      62      0.080645        4       2       0       0.643   -0.44056        0.95048 -0.46351
    22      36587154        36598081        22:36587154-36598081_APOL4      62      0.096774        12      4       0       0.3989  0.76461 0.90638 0.84358
    22      36122356        36124860        22:36122356-36124860_APOL5      62      0.1129  5       2       0       0.076266        1.9741  1.1136  1.7728
    22      36900271        36900806        22:36900271-36900806_FOXRED2    NA      NA      2       0       0       NA      NA      NA      NA
    22      36681163        36710183        22:36681163-36710183_MYH9       62      0.048387        3       1       0       0.9904  16.668  1385.4  0.012031
    22      36711990        36711990        22:36711990-36711990_Metazoa_SRP        NA      NA      1       0       0       NA      NA      NA      NA
    22      36424450        36424450        22:36424450-36424450_RBFOX2     62      0.032258        1       1       0       1       5.9095e-16      1.4376  4.1107e-16
    22      36792162        36792162        22:36792162-36792162_RP4-633O19__A.1    NA      NA      1       0       0       NA      NA      NA      NA
    

    You can run SKAT-O test in a similar way, but with a special tag

    $EPACTS/bin/epacts group --ped $SS/assoc/seqshop.ped --vcf $OUT/assoc/snps.anno.vcf.gz --out $OUT/assoc/group.skato --test skat --skat-o --groupf $OUT/assoc/snps.anno.grp --pheno PHENO --run 2
    

    And view output files

    cat $OUT/assoc/group.skato.epacts
    

    View Output file

    #CHROM	BEGIN	END	MARKER_ID	NS	FRAC_WITH_RARE	NUM_ALL_VARS	NUM_PASS_VARS	NUM_SING_VARS	PVALUE	STATRHO
    22	36655735	36661906	22:36655735-36661906_APOL1	62	0.14516	7	4	0	0.22427	1
    22	36623731	36633107	22:36623731-36633107_APOL2	62	0.064516	4	1	0	0.038657	NA
    22	36537763	36556823	22:36537763-36556823_APOL3	62	0.080645	4	2	0	0.40763	0
    22	36587154	36598081	22:36587154-36598081_APOL4	62	0.096774	12	4	0	0.55686	1
    22	36122356	36124860	22:36122356-36124860_APOL5	62	0.1129	5	2	0	0.15235	0.3
    22	36900271	36900806	22:36900271-36900806_FOXRED2	NA	NA	2	0	0	NA	NA
    22	36681163	36710183	22:36681163-36710183_MYH9	62	0.048387	3	1	0	0.075809	NA
    22	36711990	36711990	22:36711990-36711990_Metazoa_SRP	NA	NA	1	0	0	NA	NA
    22	36424450	36424450	22:36424450-36424450_RBFOX2	62	0.032258	1	1	0	1	NA
    22	36792162	36792162	22:36792162-36792162_RP4-633O19__A.1	NA	NA	1	0	0	NA	NA
    

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