# for emacs: -*- mode: sh; -*- ############################################################################## # GRCh38 patch 13 build: build basic tracks on separate database for new # sequences only (relative to hg38). ############################################################################## ############################################################################## # download or rather ln -s the patch release files (DONE - 2020-02-26 - Angie) # Note: newer assemblies use refseq releases instead of genbank, but hg38 uses genbank # so continue with that when building patches. mkdir -p /hive/data/genomes/grcH38P13/genbank cd /hive/data/genomes/grcH38P13/genbank # Releases have already been downloaded to /hive/data/outside/ncbi/genomes/. ln -s /hive/data/outside/ncbi/genomes/genbank/vertebrate_mammalian/Homo_sapiens/all_assembly_versions/GCA_000001405.28_GRCh38.p13/* . ############################################################################## # Set up fasta and agp with UCSC names (DONE - 2020-02-26 - Angie) mkdir /hive/data/genomes/grcH38P13/ucsc cd /hive/data/genomes/grcH38P13/ucsc # identify sequences not in existing genome db faCount ../genbank/GCA_000001405.28_GRCh38.p13_genomic.fna.gz \ > faCount.GRCh38.p13.txt ~/kent/src/hg/makeDb/doc/hg38/scanAssemblyReport.pl \ /hive/data/genomes/hg38/chrom.sizes \ faCount.GRCh38.p13.txt ../genbank/GCA_000001405.28_GRCh38.p13_assembly_report.txt \ | grep -w new > new.sequences.list wc -l new.sequences.list #45 new.sequences.list # Extract UCSC-named FASTA for the new sequences cut -f3 new.sequences.list > extract.new.list awk '{printf "s/%s/%s/; ", $3,$1}' new.sequences.list > genbankToUCSC.sed faSomeRecords ../genbank/GCA_000001405.28_GRCh38.p13_genomic.fna.gz extract.new.list stdout \ | sed -e 's/ .*//;' \ | sed -f genbankToUCSC.sed \ | gzip -c > grcH38P13.fa.gz faSize grcH38P13.fa.gz #14769668 bases (0 N's 14769668 real 8821498 upper 5948170 lower) in 45 sequences in 1 files #Total size: mean 328214.8 sd 802643.1 min 7309 (chr13_ML143363v1_fix) max 5500449 (chr15_ML143371v1_fix) median 192531 # Compare faSize results for whole GCA_000001405.28_GRCh38.p13_genomic.fna.gz # vs. concatenation of hg38 fasta and grcH38P13.fa.gz: faSize ../genbank/GCA_000001405.28_GRCh38.p13_genomic.fna.gz #3272116950 bases (161368694 N's 3110748256 real 1928965269 upper 1181782987 lower) in 640 sequences in 1 files #Total size: mean 5112682.7 sd 26768936.7 min 970 (KI270394.1) max 248956422 (CM000663.2) median 166743 twoBitToFa /hive/data/genomes/hg38/hg38.2bit stdout \ | faSize grcH38P13.fa.gz stdin #3272116950 bases (161368694 N's 3110748256 real 1491934681 upper 1618813575 lower) in 640 sequences in 2 files #Total size: mean 5112682.7 sd 26768936.7 min 970 (chrUn_KI270394v1) max 248956422 (chr1) median 166743 # Good, everything in GCA_000001405.28_GRCh38.p13_genomic.fna.gz is accounted for # between hg38.2bit and grcH38P13.fa.gz # Make UCSC-named AGP: zcat ../genbank/GCA_000001405.28_GRCh38.p13_assembly_structure/PATCHES/alt_scaffolds/AGP/alt.scaf.agp.gz \ | grep -Fwf extract.new.list \ | sed -f genbankToUCSC.sed > grcH38P13.agp # construct 2bit file: cd /hive/data/genomes/grcH38P13 faToTwoBit ucsc/grcH38P13.fa.gz grcH38P13.unmasked.2bit twoBitInfo grcH38P13.unmasked.2bit stdout | sort -k2nr > chrom.sizes # take a look at chrom.sizes to verify it looks OK. # Make sure AGP and FASTA/2bit agree: checkAgpAndFa ucsc/grcH38P13.agp grcH38P13.unmasked.2bit | tail -1 #All AGP and FASTA entries agree - both files are valid ############################################################################## # establish config.ra file (DONE - Angie - 2020-02-26) # arguments here are: cd /hive/data/genomes/grcH38P13 # Must make photoReference.txt first -- copy from hg38 cp /hive/data/genomes/hg38/photoReference.txt . $HOME/kent/src/hg/utils/automation/prepConfig.pl grcH38P13 haplotypes \ GRCh38.p13 genbank/*_assembly_report.txt > grcH38P13.config.ra # Edit grcH38P13.config.ra to avoid confusion with actual hg38 assemblyDate Feb. 2019 p13 orderKey 2001 sed -e 's/^/#/' grcH38P13.config.ra ## config parameters for makeGenomeDb.pl: #db grcH38P13 #clade haplotypes #scientificName Homo sapiens #commonName Human #assemblyDate Feb. 2019 p13 #assemblyLabel Genome Reference Consortium #assemblyShortLabel GRCh38.p13 #orderKey 2001 ## mitochondrial sequence included in refseq release ## mitoAcc J01415.2 #mitoAcc none #fastaFiles /hive/data/genomes/grcH38P13/ucsc/*.fa.gz #agpFiles /hive/data/genomes/grcH38P13/ucsc/*.agp ## qualFiles none #dbDbSpeciesDir GRCh38.p13 #photoCreditURL http://www.cbse.ucsc.edu/ #photoCreditName Graphic courtesy of CBSE #ncbiGenomeId 51 #ncbiAssemblyId 2334371 #ncbiAssemblyName GRCh38.p13 #ncbiBioProject 31257 #ncbiBioSample notFound #genBankAccessionID GCA_000001405.28 #taxId 9606 ############################################################################## # Initial database build (DONE - 2020-02-26 - Angie) cd /hive/data/genomes/grcH38P13 # AGP and unmasked.2bit are already built and checked, so start at the db step: mkdir jkStuff $HOME/kent/src/hg/utils/automation/makeGenomeDb.pl grcH38P13.config.ra -debug #HgStepManager: executing from step 'seq' through step 'trackDb'. #HgStepManager: executing step 'seq' Wed Feb 26 16:55:27 2020. #seq: looks like this was run successfully already (/cluster/data/grcH38P13/chrom.sizes exists). Either run with -continue agp or some later step, or move aside/remove /cluster/data/grcH38P13/chrom.sizes and run again. # Make chromInfo.tab. mkdir -p bed/chromInfo awk '{print $1 "\t" $2 "\t/gbdb/grcH38P13/grcH38P13.2bit";}' chrom.sizes \ > bed/chromInfo/chromInfo.tab # Make a link to the .agp file where makeGenomeDb.pl expects to find it. ln -s ucsc/grcH38P13.agp . # Skip 'seq' and 'agp' steps because those were done above. time ($HOME/kent/src/hg/utils/automation/makeGenomeDb.pl \ -workhorse=hgwdev -dbHost=hgwdev -fileServer=hgwdev \ -continue=db grcH38P13.config.ra) > db.log 2>&1 & tail -f db.log #real 0m22.790s # Ignore all the "NOTES -- STUFF THAT YOU WILL HAVE TO DO --" stuff because this is # going to be folded into hg38. # Now the gold, gap and gc5BaseBw tracks are built. ############################################################################# # RepeatMasker (DONE - 2020-02-27 - Angie) mkdir /hive/data/genomes/grcH38P13/bed/repeatMasker cd /hive/data/genomes/grcH38P13/bed/repeatMasker time ($HOME/kent/src/hg/utils/automation/doRepeatMasker.pl -buildDir=`pwd` \ -bigClusterHub=ku -dbHost=hgwdev -workhorse=hgwdev \ -smallClusterHub=ku grcH38P13) > do.log 2>&1 & tail -f do.log #real 58m13.702s egrep "bases|Total|masked" faSize.rmsk.txt \ | fold -w 95 -s | sed -e 's/^/# /;' # 14769668 bases (0 N's 14769668 real 6559203 upper 8210465 lower) in 45 sequences in 1 files # Total size: mean 328214.8 sd 802643.1 min 7309 (chr13_ML143363v1_fix) max 5500449 # (chr15_ML143371v1_fix) median 192531 # %55.59 masked total, %55.59 masked real egrep -i "versi|relea" do.log | sed -e 's/^/# /;' # RepeatMasker version development-$Id: RepeatMasker,v 1.332 2017/04/17 19:01:11 rhubley Exp $ # grep version of RepeatMasker$ /hive/data/staging/data/RepeatMasker/RepeatMasker # # February 01 2017 (open-4-0-8) 1.332 version of RepeatMasker # grep RELEASE /hive/data/staging/data/RepeatMasker/Libraries/RepeatMaskerLib.embl # CC Dfam_Consensus RELEASE 20181026; * # CC RepBase RELEASE 20181026; * featureBits -countGaps grcH38P13 rmsk #8210465 bases of 14769668 (55.590%) in intersection ########################################################################## # running simple repeat (DONE - 2020-02-27 - Angie) mkdir /hive/data/genomes/grcH38P13/bed/simpleRepeat cd /hive/data/genomes/grcH38P13/bed/simpleRepeat # using trf409 6 here like hg38 time ($HOME/kent/src/hg/utils/automation/doSimpleRepeat.pl -buildDir=`pwd` \ -dbHost=hgwdev -workhorse=hgwdev -bigClusterHub=ku -smallClusterHub=ku \ -trf409 6 grcH38P13) > do.log 2>&1 & #real 6m49.141s cat fb.simpleRepeat #1549326 bases of 14769668 (10.490%) in intersection # adding this trfMask to .rmsk.2bit cd /hive/data/genomes/grcH38P13 twoBitMask grcH38P13.rmsk.2bit \ -add bed/simpleRepeat/trfMask.bed grcH38P13.2bit # you can safely ignore the warning about fields >= 13 twoBitToFa grcH38P13.2bit stdout | faSize stdin > faSize.grcH38P13.2bit.txt egrep "bases|Total|masked" faSize.grcH38P13.2bit.txt \ | fold -w 95 -s | sed -e 's/^/# /;' # 14769668 bases (0 N's 14769668 real 6528429 upper 8241239 lower) in 45 sequences in 1 files # Total size: mean 328214.8 sd 802643.1 min 7309 (chr13_ML143363v1_fix) max 5500449 # (chr15_ML143371v1_fix) median 192531 # %55.80 masked total, %55.80 masked real # reset the symlink ln -sf `pwd`/grcH38P13.2bit /gbdb/grcH38P13/grcH38P13.2bit ########################################################################## ## WINDOWMASKER (DONE - 2020-02-27 - Angie) mkdir /hive/data/genomes/grcH38P13/bed/windowMasker cd /hive/data/genomes/grcH38P13/bed/windowMasker time ($HOME/kent/src/hg/utils/automation/doWindowMasker.pl -buildDir=`pwd` \ -workhorse=hgwdev -dbHost=hgwdev grcH38P13) > do.log 2>&1 # *** All done ! - Elapsed time: 0m47s featureBits -countGaps grcH38P13 rmsk windowmaskerSdust \ > fb.grcH38P13.rmsk.windowmaskerSdust.txt 2>&1 cat fb.grcH38P13.rmsk.windowmaskerSdust.txt #3364201 bases of 14769668 (22.778%) in intersection # Masking statistics egrep "bases|Total|masked" faSize.grcH38P13.cleanWMSdust.txt \ | fold -w 95 -s | sed -e 's/^/# /;' # 14769668 bases (0 N's 14769668 real 10765455 upper 4004213 lower) in 45 sequences in 1 files # Total size: mean 328214.8 sd 802643.1 min 7309 (chr13_ML143363v1_fix) max 5500449 # (chr15_ML143371v1_fix) median 192531 # %27.11 masked total, %27.11 masked real ############################################################################# # cytoBandIdeo - (DONE - 2020-02-27 - Angie) mkdir /hive/data/genomes/grcH38P13/bed/cytoBand cd /hive/data/genomes/grcH38P13/bed/cytoBand makeCytoBandIdeo.csh grcH38P13 ############################################################################# # cpgIslands - (DONE - 2020-02-27 - Angie) mkdir /hive/data/genomes/grcH38P13/bed/cpgIslands cd /hive/data/genomes/grcH38P13/bed/cpgIslands time ($HOME/kent/src/hg/utils/automation/doCpgIslands.pl -dbHost=hgwdev \ -workhorse=hgwdev -smallClusterHub=ku -bigClusterHub=ku grcH38P13) > do.log 2>&1 & # *** All done ! Elapsed time: 1m10s cat fb.grcH38P13.cpgIslandExt.txt #232897 bases of 14769668 (1.577%) in intersection ############################################################################## # genscan - (DONE - 2020-02-27 - Angie) mkdir /hive/data/genomes/grcH38P13/bed/genscan cd /hive/data/genomes/grcH38P13/bed/genscan time ($HOME/kent/src/hg/utils/automation/doGenscan.pl -buildDir=`pwd` \ -workhorse=hgwdev -dbHost=hgwdev -bigClusterHub=ku grcH38P13) > do.log 2>&1 & # *** All done ! Elapsed time: 3m11s cat fb.grcH38P13.genscan.txt #478205 bases of 14769668 (3.238%) in intersection cat fb.grcH38P13.genscanSubopt.txt #442575 bases of 14769668 (2.997%) in intersection ############################################################################# # augustus gene track (DONE - 2020-02-27 - Angie) mkdir /hive/data/genomes/grcH38P13/bed/augustus cd /hive/data/genomes/grcH38P13/bed/augustus time ($HOME/kent/src/hg/utils/automation/doAugustus.pl -buildDir=`pwd` -bigClusterHub=ku \ -species=human -dbHost=hgwdev -workhorse=hgwdev grcH38P13) > do.log 2>&1 & # *** All done ! Elapsed time: 30m26s cat fb.grcH38P13.augustusGene.txt #418904 bases of 14769668 (2.836%) in intersection ############################################################################## # Download files (DONE - 2020-02-27 - Angie) cd /hive/data/genomes/grcH38P13 time ($HOME/kent/src/hg/utils/automation/makeDownloads.pl -noChromFiles \ -workhorse=hgwdev grcH38P13) > downloads.log 2>&1 & # *** All done! #real 0m20.430s ############################################################################## # PREPARE LINEAGE SPECIFIC REPEAT FILES FOR LASTZ (DONE - 2020-02-27 - Angie) mkdir /hive/data/genomes/grcH38P13/bed/linSpecRep cd /hive/data/genomes/grcH38P13/bed/linSpecRep # create individual .out files from the master record in ../repeatMasker # cluster job is perhaps overkill for just the patches, but it works mkdir splitOut cat <<'_EOF_' > split.csh #!/bin/csh -fe set C = $1 head -3 ../repeatMasker/grcH38P13.sorted.fa.out > splitOut/${C}.out grep "${C} " ../repeatMasker/grcH38P13.sorted.fa.out >> splitOut/${C}.out _EOF_ chmod +x split.csh cat << '_EOF_' > template #LOOP split.csh $(root1) {check out line+ splitOut/$(root1).out} #ENDLOOP _EOF_ # small ones first: cut -f1 ../../chrom.sizes | tac > chrom.list gensub2 chrom.list single template jobList para make jobList para time #Completed: 45 of 45 jobs #CPU time in finished jobs: 1s 0.01m 0.00h 0.00d 0.000 y #IO & Wait Time: 132s 2.21m 0.04h 0.00d 0.000 y #Average job time: 3s 0.05m 0.00h 0.00d #Longest finished job: 5s 0.08m 0.00h 0.00d #Submission to last job: 20s 0.33m 0.01h 0.00d # now, we can date and process each of those .out files # constructing the humanSpecific set of repeats # this means repeats found in human, and not in others # using mouse here for 'others' is good enough, a variety # of other species could be used (rat dog cow) where they all # produce the same result mkdir dateRepeats cd dateRepeats cat << '_EOF_' > mkLSR #!/bin/bash set -beEu -o pipefail seq=$1 rm -f $seq.out_mus-musculus ln -sf ../splitOut/$seq.out . /scratch/data/RepeatMasker/DateRepeats $seq.out -query human -comp 'mus musculus' rm $seq.out mkdir -p ../humanSpecific /cluster/bin/scripts/extractRepeats 1 $seq.out_mus-musculus \ > ../humanSpecific/$seq.out.spec _EOF_ chmod +x mkLSR cat << '_EOF_' > template #LOOP ./mkLSR $(path1) {check out line+ ../humanSpecific/$(path1).out.spec} #ENDLOOP _EOF_ gensub2 ../chrom.list single template jobList para make jobList para time #Completed: 45 of 45 jobs #CPU time in finished jobs: 893s 14.88m 0.25h 0.01d 0.000 y #IO & Wait Time: 95s 1.59m 0.03h 0.00d 0.000 y #Average job time: 22s 0.37m 0.01h 0.00d #Longest finished job: 26s 0.43m 0.01h 0.00d #Submission to last job: 59s 0.98m 0.02h 0.00d # We also need the nibs for blastz runs with lineage specific repeats mkdir /hive/data/genomes/grcH38P13/bed/nibs cd /hive/data/genomes/grcH38P13/bed/nibs cut -f1 ../../chrom.sizes | while read C do twoBitToFa -seq=${C} ../../grcH38P13.2bit stdout \ | faToNib -softMask stdin ${C}.nib echo "${C} done" done # verify nothing lost cat ../../chrom.sizes \ | awk '{printf "nibFrag -masked %s.nib 0 %d + stdout\n", $1, $2}' \ | sh | faSize stdin #14769668 bases (0 N's 14769668 real 6528429 upper 8241239 lower) in 45 sequences in 1 files #Total size: mean 328214.8 sd 802643.1 min 7309 (chr13_ML143363v1_fix.nib:0-7309) max 5500449 (chr15_ML143371v1_fix.nib:0-5500449) median 192531 #N count: mean 0.0 sd 0.0 #U count: mean 145076.2 sd 355830.6 #L count: mean 183138.6 sd 450022.5 #%55.80 masked total, %55.80 masked real mkdir -p /hive/data/staging/data/grcH38P13/nib rsync -a --progress ./ /hive/data/staging/data/grcH38P13/nib rsync -a --progress /hive/data/genomes/grcH38P13/{grcH38P13.2bit,chrom.sizes} \ /hive/data/staging/data/grcH38P13/ ##############################################################################