View source for SeqShop: Ancestry On Your Own Genome, December 2014

__TOC__

== Setup ==
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''If you are already logged in, you can skip this section.''
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{{SeqShopLogin}}
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== Setup your terminal ==

Resume screen:
 screen -r


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

While that's running, let's go run the Association Analysis Practical: [[SeqShop: Genetic Association Analysis Practical, December 2014]]

=== Checking if Pileup finished ===
 screen -r

'''Did everyone's finish?'''


If not, let's go to Indel: [[SeqShop:_Calling_Your_Own_Genome,_December_2014#Reviewing_Indel_Results]]


If yes, let's continue below:
=== 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

== Looking at Calling results ==
If we haven't done INDEL: [[]]

If we are moving on to SNPs: [[]]

== 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).

[[File:HGDP Popualtions.png|thumb|center|alt=HGDP populations |300px|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 [[SeqShop:_Calling_Your_Own_Genome,_December_2014#Reviewing_Indel_Results]] and pick up where we left off.