Dpuiu CA

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 cvs -z3 -d:pserver:anonymous@wgs-assembler.cvs.sourceforge.net:/cvsroot/wgs-assembler co -P wgs-assembler

• Compile for debugging

 BUILDDEBUG  = 1

Run

• Help

runCA.pl -fields

• Batch submission

 touch runCA.log
   echo --- runCA $$ restarted by $USER at `date` --- >>& runCA.log
   runCA -d . -p prefix -s spec prefix.frg >>& runCA.log

 at runCA.sh   

• Sanger

runCA -d . -p prefix -s spec prefix.frg

• doOverlapTrimming=1 (default)
• low qual reads (ex: 20 or 'D') are discarded
• astatLowBound 1 (default)  ; can be lowered for low/un-uniform coverage
• astatHighBound 5 (default)
• computeInsertSize=0 (default)

• 454 & Hybrid

runCA -d . overlapper=mer unitigger=bog -p prefix *frg *.sff

• gatekeeper -L : search for 454 paired end linker
• AS_GKP_sff.c  : Load_SFF function
• for mated libs set mean=3000,stdev=300

Parameters

• stopAfter:

   initialStoreBuilding
   overlapBasedTrimming | OBT
   overlapper
   unitigger
   consensusAfterUnitigger
   scaffolder
   consensusAfterScaffolder

• cgwOutputIntermediate = 1 => "cgw -G"

 => .cgw & .cgw_contigs (generated only at the last doExtendClearRanges step)

• doExtendClearRanges = 2 => cgw run 3 times, ECR 2 times

Gatekeeper

Update

• Using messages : act:R !!!

 {DST
 act:R
 acc:10691
 mea:161662
 std:28760
 }

• Using tab file

 Example: No restrictions on lib stddev with --edit option

 cat prefix.update
 lib     uid     19070   mean    167001
 lib     uid     19070   stddev  167001

 gatekeeper --edit prefix.update prefix.gkpStore > prefix.gkpStore.seq

Delete

• DST messages can not be deleted
• A FRG can be deleted only if there is no related LKG message; an abbreviated MSG

 {LKG
 act:D
 typ:M
 fg1:94376
 fg2:94654
 }
 {FRG
 act:D
 acc:94376
 }

Dump

• sequences

 gatekeeper -dumpfragments -withsequence prefix.gkpStore > prefix.gkpStore.seq

• frg file

 gatekeeper -dumpfrg prefix.gkpStore > prefix.gkpStore.frg

• all

 gatekeeper -dumpfragments -tabular prefix.gkpStore > prefix.gkpStore.tab

• clear ranges (latest=ECR2)

 gatekeeper -dumpfragments -tabular -clear [ORIG|QLT|VEC|OBTINI|OBT|UTG|ECR1|ECR2] prefix.gkpStore/  | perl -ane 'print join "\t", @F[0,-2,-1]; print "\n";' > prefix.ECR2.clr

Log file & runtime (wgs-5.2)

• Use ~dpuiu/bin/getCAruntimes.pl
• runCA.log runCA.times bos taurus BCM.CLONEEND 16,875 Sanger reads
• runCA.log runCA.times wolbachia symbioint of culex pipiens quinquefasciatus 36,767 Sanger reads

Output processing

Posmap

• Get posMap files

buildPosMap -o prefix < prefix.asm

• Get number of reads/ctg or scf (from the pos files)

 cat prefix.posmap.frgctg | count.pl > prefix.posmap.frgctg.count
 cat prefix.posmap.frgctg | count.pl > prefix.posmap.frgscf.count
 cat prefix.posmap.ctgscf | count.pl > prefix.posmap.ctgscf.count
 ...

• Compute read/mate/ctg cvg (from the pos files)

 posmap2cvg.pl                            < prefix.posmap.frgscf > prefix.posmap.frgscf.frg_cvg
 posmap2cvg.pl -mates prefix.posmap.mates < prefix.posmap.frgscf > prefix.posmap.frgscf.mate_cvg
 posmap2cvg.pl                             < prefix.posmap.ctgscf > prefix.posmap.ctg_cvg

• Qc stats based on posmap files

 posmap2qc.pl -ctgscf prefix.posmap.ctgscf > prefix.qc

• Break scf/ctg pipeline

 ~/bin/breakPosmapKeep.amos

• Add ctg/scf into to the mates fils

 join12.pl prefix.posmap.mates prefix.posmap.frgctg > prefix.posmap.mates.ctg2
 join12.pl prefix.posmap.mates.ctg2 prefix.posmap.frgscf > prefix.posmap.mates.ctg2.scf2

• Get ctg/deg/scaffold links from the posmap files

 ~/bin/join12.pl -0 prefix.posmap.mates prefix.posmap.frgscf prefix.posmap.frgdeg >! prefix.posmap.mates2
 cat prefix.posmap.mates2 | awk '{print $3,$4,$8}' | p '@F[1,2]=@F[2,1] if ($F[1] gt $F[2]); print join  "\t",@F; print "\n"' | count.pl >  prefix.posmap.mates2.count
 cat prefix.posmap.mates2.count | grep -v '\.' | p 'print $_ if($F[1] ne $F[2]);' > prefix.posmap.mates2.count.diff
  
 cat prefix.posmap.mates2.count.diff | grep diffScaff | cut -f2,3,4 | ~/bin/cluster.pl > ! prefix.posmap.mates2.count.diffScaff.cluster
 cat prefix.posmap.mates2.7180002040716-7180002040985 | perl -ane '@F[0,1, 3,4,5,6, 7,8,9,10 ]=@F[1,0, 7,8,9,10,  3,4,5,6, ] if ($F[3] gt $F[7]); print join "\t",@F; print "\n";' | awk '{print $7,$11}' | count.pl

• Display graphs

Fasta

• Align deg to scaff

 nucmer -mum -l 40 -c 250 scf.fasta deg.fasta  -p scf-deg
 delta-filter -q scf-deg.delta > scf-deg.filter-q.delta
 ~/bin/delta2pairCvg+.pl < scf-deg.filter-q.delta > scf-deg.filter-q.cvg.pairs
 ~/bin/max2.pl -i 4 -j 6 < scf-deg.filter-q.cvg.pairs > scf-deg.filter-q.maxCvg.pairs
 cat scf-deg.filter-q.maxCvg.pairs | p 'print $_ if($F[5]-$F[6]<2000);' > scf-deg.filter-q.variants.pairs
 awk '{print $5,$6}' scf-deg.filter-q.variants.pairs > scf-deg.filter-q.variants.ids

• Indexing

 # contigs
 ~/bin/formatdb.pl < prefix.ctg.fasta > prefix.ctg.fasta.offset
 ~/bin/fastacmd.pl -o prefix.ctg.fasta.offset -f filter.ctg.ids < prefix.ctg.fasta > filter.ctg.fasta

 # same for scaff, deg ...

• Contaminant search : nucmer against

 /nfshomes/dpuiu/db/Ecoli.all
 /nfshomes/dpuiu/db/UniVec_Core
 /nfshomes/dpuiu/db/OtherVec

Asm

• Get output seq

 asmOutputFasta -p prefix < prefix.asm

• Get output seq clr

 ~/bin/asm2clrs.pl < prefix.asm > < prefix.asm.clr

• Get new library insert sizes:

 asm2mdi.pl < prefix.asm > prefix.mdi

• Get surrogate ids

 #most of them have negative aStat's
 $ cat *.asm | grep -B 8 ^sta:S | grep ^acc | perl -ane '/(\d+)/; print "utg".$1,"\n";'

Scaffolds

• Get scaff files

 cavalidate -e 10 prefix
 bank2scaff prefix.bnk/ > prefix.scaff

 ~/bin/ca2scaff -i prefix.asm -o prefix.scaff

 cat prefix.scaff | grep "^>" | sed 's/>//' | awk '{print $1,$2}' > prefix.scfctg
 cat prefix.scaff | grep "^>" | sed 's/>//' | awk '{print $1,$3}' > prefix.scflen  
 cat prefix.scaff | grep "^>" | sed 's/>//' | awk '{print $1,$4}' > prefix.scfspan  

 tac prefix.scaff | p '$count=0 if(/^>/); $count++; print $_ if($count>2);' | awk '{print $4}'   # sequence gaps

AGP

 asmToAGP.pl < prefix.asm > prefix.agp

        /--- buildPosMap ------> .posmap
       /
  .asm /---- asmToAGP ------------------------------------------------> .AGP 
      \                                                              /   
       \---- cavalidate, bank2scaff?--> .scaff --- scaff2agp.pl --- 

• Other scripts:

 bank2scaff : incorrect gap sizes

 valiadteAgp.pl < prefix.agp

 infoseq 9-terminator/prefix.ctg.fasta | sed 's/ctg//' >! prefix.ctg.infoseq
 scaff2agp.pl -i prefix.ctg.infoseq < prefix.scaff > prefix.agp

Running subassemblies

Update frg file:

• clr=ECR2
• MDI -> DST

runCA parameters:

• doOBT=no

Test datasets

Porphyromonas_gingivalis_W83 : Sanger + 454

• TA  : ftp://ftp.ncbi.nih.gov/pub/TraceDB/porphyromonas_gingivalis_w83
• SRA : http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=Link&LinkName=genomeprj_sra&from_uid=29961