SeqShop: Ancestry On Your Own Genome, December 2014

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Setup

If you are already logged in, you can skip 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
  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

      • Xming.png

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

      • PuttyS.png

  4. 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
  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

Setup your terminal

Resume screen: 

screen -r


Only if your snpcalling is still running, let Mary Kate know, expand, and follow these instructions

  • Detach from screen
Ctrl-a d
  • Start new screen
screen
  • Reset environment variables (replace SampleXX with your sample name)
export SAMPLE=SampleXX
source /net/seqshop-server/home/mktrost/seqshop/setupSS.txt
  • When you detach from screen and reattach, you will have multiple screen sessions
    • The ancestry one will have today's date, the snpcall one will have an earlier date.
      • Mary Kate can explain how to reattach to a specific session when we get there.


Instructions for everyone: Setup LASER

Source the LASER setup: 

source /net/seqshop-server/home/chaolong/LASER-Tutorial/setup.txt

(SAMPLE & OUT were previously set in your screen session.)

After setting this, also do 

mkdir -p $OUT/ancestry

Verify that this does not give an error: 

ls $OUT/bams/${SAMPLE}.recal.bam

Run

Step 1: bam --> pileup

$GC/bin/samtools mpileup -q 30 -Q 20 -f $REF/human.g1k.v37.fa -l $HGDP/HGDP_938.bed $OUT/bams/${SAMPLE}.recal.bam > $OUT/ancestry/${SAMPLE}.HGDP.pileup

This step takes ~1 hour for a genome sequenced at 17X.

While this is running, we will go look at other results, so exit screen: 

Ctrl-a d

Let's go run the Association Analysis Practical: SeqShop: Genetic Association Analysis Practical, December 2014

Step 2: pileup --> seq

python $LASER/pileup2seq/pileup2seq.py \
-m $HGDP/HGDP_938.site \
-o $OUT/ancestry/$SAMPLE.HGDP \
$OUT/ancestry/$SAMPLE.HGDP.pileup

This step takes just a few seconds.

Estimate ancestry

This step will take about 5-6 minutes. 

$LASER/laser -g $HGDP/HGDP_938.geno -c $HGDP/HGDP_938.RefPC.coord -s $OUT/ancestry/$SAMPLE.HGDP.seq -K 20 -k 4 -M 0.8 -o $OUT/ancestry/$SAMPLE.HGDP &

View the results: 

less -S $OUT/ancestry/${SAMPLE}.HGDP.SeqPC.coord

Visualizing Ancestry

Copy the R code to plot your ancestry 

cp -r $LASER/plot/ $OUT/ancestry/.

Change to that new directory: 

cd $OUT/ancestry/plot

Generate the plot: 

Rscript plotHGDP.r $HGDP/HGDP_938.RefPC.coord $OUT/ancestry/${SAMPLE}.HGDP.SeqPC.coord

Take a look: 

evince Results_on_HGDP.pdf &

Interested in looking just at European or East Asian or Central South Asian populations?

Population information

8 European populations in the HGDP dataset, including 156 individuals in total.

18 East Asian populations in the HGDP dataset, including 224 individuals after excluding 5 outliers (defined as more than 5 standard deviations from mean in any of top 10 PCs, 1 removal iteration).

8 Central/South Asian populations in the HGDP dataset, including 195 individuals after excluding 5 outliers (defined as more than 5 standard deviations from mean in any of top 10 PCs, 1 removal iteration). Note that 7 out of these 8 population were collected in Pakistan and 1 was collected in China (Uygur).

HGDP populations
HGDP populations

Step 1: bam --> pileup

You can skip this, you already did it.

Step 2: pileup --> seq

You can skip this step too, because you already did it.

Estimate ancestry

This step will take a few seconds.

For European ancestry: 

$LASER/laser -g $HGDP/HGDP.633K.euro.geno -c $HGDP/HGDP.633K.euro.RefPC.coord -s $OUT/ancestry/$SAMPLE.HGDP.seq -K 20 -k 4 -M 0.8 -o $OUT/ancestry/$SAMPLE.HGDP-Euro &

For East Asian ancestry: 

$LASER/laser -g $HGDP/HGDP.633K.easia.2.geno -c $HGDP/HGDP.633K.easia.2.RefPC.coord -s $OUT/ancestry/$SAMPLE.HGDP.seq -K 20 -k 4 -M 0.8 -o $OUT/ancestry/$SAMPLE.HGDP-EAsia &

For Central/South Asian ancestry: 

$LASER/laser -g $HGDP/HGDP.633K.csasia.2.geno -c $HGDP/HGDP.633K.csasia.2.RefPC.coord -s $OUT/ancestry/$SAMPLE.HGDP.seq -K 20 -k 4 -M 0.8 -o $OUT/ancestry/$SAMPLE.HGDP-CSAsia &


View the results for European ancestry: 

less -S $OUT/ancestry/${SAMPLE}.HGDP-Euro.SeqPC.coord

or for East Asian ancestry 

less -S $OUT/ancestry/${SAMPLE}.HGDP-EAsia.SeqPC.coord

or for Central/South Asian ancestry 

less -S $OUT/ancestry/${SAMPLE}.HGDP-CSAsia.SeqPC.coord

Visualizing Ancestry

Copy the R code to plot your ancestry 

cp -r $LASER/plot/ $OUT/ancestry/.

Change to that new directory: 

cd $OUT/ancestry/plot

Move your other plot so you don't over-write it 

mv Results_on_HGDP.pdf Results_on_HGDP_All.pdf


Generate the plot for European ancestry: 

Rscript plotHGDP.r $HGDP/HGDP.633K.euro.RefPC.coord $OUT/ancestry/${SAMPLE}.HGDP-Euro.SeqPC.coord

or for East Asian ancestry 

Rscript plotHGDP.r $HGDP/HGDP.633K.easia.2.RefPC.coord $OUT/ancestry/${SAMPLE}.HGDP-EAsia.SeqPC.coord

or for Central/South Asian ancestry 

Rscript plotHGDP.r $HGDP/HGDP.633K.csasia.2.RefPC.coord $OUT/ancestry/${SAMPLE}.HGDP-CSAsia.SeqPC.coord

Take a look: 

evince Results_on_HGDP.pdf &


Return to Calling your own Genome

Go to Reviewing SNPCALL Results and pick up where we left off.

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