PROVEAN'ın yüklenmesi ve çalıştırılması ile protein varyantlarının etkisinin tahmini

PROVEAN girdi dosyaları, aşağıdaki komutlar kullanılarak VEP çıktısına göre oluşturuldu. PROVEAN'ın tüm açıklamalı genlerde paralel olarak ne kadar hızlı çalıştırılabileceğini anlamak için bazı deneme çalıştırmaları bir bilgisayarda tamamlandı.

# running PROVEAN

# installation & dependencies
# 1. checked that blast was installed and also reinstalled cd-hit to avoid issue with certain version
# 2. installed the NCBI nr database
sudo mkdir /opt/ncbi_blast_nr_db_2018-01-29
sudo chmod 775 /opt/ncbi_blast_nr_db_2018-01-29
sudo chown administrator:administrator /opt/ncbi_blast_nr_db_2018-01-29
cd /opt/ncbi_blast_nr_db_2018-01-29
update_blastdb --passive --decompress --timeout 300 --force --verbose nr
# 3. install provean
tar xvf provean-1.1.5.tar.gz
cd provean-1.1.5
./configure BLAST_DB=/opt/ncbi_blast_nr_db_2018-01-29/nr
make
sudo make install

# prepare PROVEAN inputs using protein sequence fasta and the results of a VEP analysis
# create bed summary of output that we can easily do random searches from
cat Boco_wgs_BZ_HN_joint_variants_VEP_effects | \
awk '{ if ($11 ~ "/") print $0 }' | cut -f 5,10,11 | \
awk -v OFS="\t" '{ print $1, $2+0, $2+1, $3 }' | \
sort -k1,1 -k2,2n | bgzip -c \
> Boco_wgs_BZ_HN_joint_variants_VEP_effects.coding.bed.gz

# index this bed and the protein fasta
tabix -p bed Boco_wgs_BZ_HN_joint_variants_VEP_effects.coding.bed.gz
samtools faidx Bcon_rnd3.all.maker.proteins.fasta

# pull out protein variants and format properly, and gather protein fasta sequences
# variants with an X (any amino acid) are excluded
cat Boco_wgs_BZ_HN_joint_variants_VEP_effects | \
awk '{ if ($11 ~ "/") print $0 }' | cut -f5 | sort | uniq | \
while read protein; \
do \
echo ${protein}; \
tabix Boco_wgs_BZ_HN_joint_variants_VEP_effects.coding.bed.gz ${protein} | \
awk -v OFS="\t" '{ if ($4 !~ "X") print $0 }' | \
awk -F "\t|/" -v OFS="," '{ print $2, $4, $5 }' | sed 's/-/./g' \
> input_files_per_protein/${protein}.var.txt; \
samtools faidx Bcon_rnd3.all.maker.proteins.fasta ${protein} \
> input_files_per_protein/${protein}.seq.fasta; \
done

# I am able to run PROVEAN just fine, but it takes N minutes on 1 core for a protein 
# of average length, mostly due to the BLAST. Given we need to run almost 15k proteins,
# we need to parallelize this a lot. I plan to run 4 sets of jobs in parallel on 
# moonunits 0-2 and javelina (2000 proteins each, running on 10 cores, total run time
# of 8-10 days). I will also run 1 large set of jobs using jetstream (8000 proteins, 
# running on 43 cores, total run time of 8-9 days). Math assumes 1 core per job and that
# jobs last approximately N minutes on average.