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popoolation.md

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workfow for the population genetic analyses

Make mpileup

Use the final bam files, using merged files for the artifical selection and unmerged files for wild

make mpileup script

Masking Repeats

Used the repetitive regions identified by RepeatMasker and the script from Popoolation2 (v1.2.2)

perl /home/katie/bin/popoolation_1.2.2/basic-pipeline/filter-pileup-by-gtf.pl --gtf /home/katie/flyGenome/dmel_r6.23/dmel-all-chromosome-r6.23.fasta.out.gff --input ee_merge.mpileup --output ee_merge_repeatmask.mpileup &
perl /home/katie/bin/popoolation_1.2.2/basic-pipeline/filter-pileup-by-gtf.pl --gtf /home/katie/flyGenome/dmel_r6.23/dmel-all-chromosome-r6.23.fasta.out.gff --input wild_nomerge.mpileup --output wild_nomerge_repeatmask.mpileup

Masking InDels

First I identified indels using the popoolation2 script, used a 5bp window around indels

perl /home/katie/bin/popoolation2_1201/indel_filtering/identify-indel-regions.pl --input ee_merge.mpileup --output ee_merge_indels.gtf --indel-window 5 &
perl /home/katie/bin/popoolation2_1201/indel_filtering/identify-indel-regions.pl --input wild_nomerge.mpileup --output wild_nomerge_indels.gtf --indel-window 5

Then mask indels in mpileup file

perl /home/katie/bin/popoolation_1.2.2/basic-pipeline/filter-pileup-by-gtf.pl --gtf ee_merge_indels.gtf --input ee_merge_repeatmask.mpileup --output ee_merge_repeatmask_indelmask.mpileup &
perl /home/katie/bin/popoolation_1.2.2/basic-pipeline/filter-pileup-by-gtf.pl --gtf wild_nomerge_indels.gtf --input wild_nomerge_repeatmask.mpileup --output wild_nomerge_repeatmask_indelmask.mpileup

Make sync file

java -ea -Xmx32g -jar /usr/local/popoolation/mpileup2sync.jar --input ee_merge_repeatmask_indelmask.mpileup --output ee_masked_merged.sync &
java -ea -Xmx32g -jar /usr/local/popoolation/mpileup2sync.jar --input wild_nomerge_repeatmask_indelmask.mpileup --output wild_masked_nomerge.sync

Calculate Fst in sliding windows

perl /home/katie/bin/popoolation2_1201/fst-sliding.pl --input ee_masked_merged.sync --output ../population/ee_merged_5000windows.fst --suppress-noninformative --min-count 2 --min-coverage 10 --max-coverage 600 --min-covered-fraction 0.2 --window-size 5000 --step-size 5000 --pool-size 75 &
perl /home/katie/bin/popoolation2_1201/fst-sliding.pl --input wild_masked_nomerge.sync --output ../population/wild_nomerge_100windows.fst --suppress-noninformative --min-count 2 --min-coverage 10 --max-coverage 600 --min-covered-fraction 0.2 --window-size 100 --step-size 5000 --pool-size 75

For the wild populations, I want to pull out only the comparisons I am interested in.

awk '{print $1, $2, $3, $4, $5, $6, $36, $57, $71}' wild_nosync_100window.fst > wild_lvr_nosync_100window.fst &
awk '{print $1, $2, $3, $4, $5, $7, $37, $58, $69}' wild_nosync_100window.fst > wild_lvc_nosync_100window.fst &
awk '{print $1, $2, $3, $4, $5, $17, $44, $62, $70}' wild_nosync_100window.fst > wild_rvc_nosync_100window.fst

For the artifical selection population the order of comparisons are: control-vs-down, control-vs-up, down-vs-up.