Revision as of 03:11, 9 March 2010

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WGS

Download & compile CVS tip

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

 cd wgs-assembler/src
 make SITE_NAME=LOCAL 

 => executables under wgs-assembler/Linux-amd64/bin/

Download & compile release

 wget ...

 bzip2 -dc wgs-6.0-beta.tar.bz2 | tar -xf -
 cd wgs-6.0-beta
 cd kmer
 sh configure.sh
 gmake install
 cd ../src
 gmake
 cd ..

Compile for debugging :

 make SITE_NAME=LOCAL  BUILDDEBUG=1  # in "c_make.as"

Run

Help

runCA.pl -fields

Batch submission

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

 at runCA.sh

Sanger

 runCA -d . -p prefix -s spec Sanger.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 : convert sff to frg

 sffToCA -libraryname 454 -output 454 454.sff
 runCA -d . ovlOverlapper=mer unitigger=bog -p prefix *.frg
 runCA -d . -s ~/bin/runCA.454.spec -p prefix *.frg

run concurently

 cnsConcurrency=4 frgCorrConcurrency=4 merOverlapperExtendConcurrency=4 merOverlapperSeedConcurrency=4 ovlConcurrency=4 ovlCorrConcurrency=4 ...

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

 Example:
 ~/bin/runCA -d . -p asm -s ~/bin/runCA.454.spec stopAfter=overlapper Saureus.frg

cgwOutputIntermediate = 1 => "cgw -G"

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

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

Gatekeeper

Build

Create & add

 gatekeeper    -o prefix.gkpStore -T -F prefix*frg
 gatekeeper -a -o prefix.gkpStore -T -F prefix*frg

Update

edit frg: act:R !!!

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

append

 gatekeeper -a -o prefix.gkpStore -T -F edit.frg

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
 lib     uid     19070   isNotRandom     1
 lib     uid     19070   Orientation     O            # outie orientation for Illimina libs >1K
    
 gatekeeper --edit prefix.update prefix.gkpStore

Update read CLR/CLV

 gatekeeper -a -r prefix.clr -o prefix.gkpStore   # not in the wgs release
 gatekeeper -a -v prefix.clv -o prefix.gkpStore

Delete

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

 # frg1
 {LKG
 act:D
 typ:M
 fg1:94376
 fg2:94654
 }
 {FRG
 act:D
 acc:94376
 }

 # frg2 ???
 {LKG
 act:D
 fg:94376
 fg:94654
 }
 {FRG
 act:D
 acc:94376
 }

cat delete.ids | perl -ane 'print "{FRG\nact:D\nacc:$F[0]\n}\n";'> delete.frg
gatekeeper -a -o prefix.gkpStore -T -F delete.frg

Dump

INFO

 gatekeeper -dumpinfo prefix.gkpStore

LAST frg in store

 gatekeeper -dumpinfo -lastfragiid prefix.gkpStore

FASTA sequences

 gatekeeper -dumpfastaseq prefix.gkpStore > prefix.seq
 gatekeeper -dumpfastaqlt prefix.gkpStore > prefix.qual

FASTA reads for newbler

 gatekeeper -dumpnewbler newbler prefix.gkpStore => prefix.fna,prefix.fna.qual

 grep "^>" prefix.fna | head -2
 >SRR001355.3635.1a template=2020+2021 dir=F library=SRX000348 trim=20-117
 >SRR001355.3635.1b template=2020+2021 dir=R library=SRX000348 trim=1-130

FRG file

 gatekeeper -dumpfrg prefix.gkpStore > prefix.frg
 gatekeeper -dumpfrg prefix.gkpStore -format2 > prefix.frg2

READS in TAB format

 gatekeeper -dumpfragments -tabular prefix.gkpStore > prefix.gkpStore.info
 gatekeeper -dumpfragments -tabular prefix.gkpStore | grep -v ^UID | awk '{print $1,$2}' > prefix.gkpStore.uid2iid

LIBS in TAB format

 gatekeeper -dumplibraries -tabular prefix.gkpStore | grep -v ^UID | awk '{print $1,$4,$5}' | sed 's/\.000//g'> prefix.dst  # original vals
 asm2mdi.pl < prefix.asm > prefix.mdi                                                                                       # final vals

READ CLR's (default=ECR2)

 gatekeeper -dumpfragments -tabular prefix.gkpStore/  | perl -ane 'print join "\t", @F[0,-2,-1]; print "\n";' > prefix.clr
 gatekeeper -dumpfragments -tabular -clear [CLR|OBTINITIAL|OBTMERGE|OBTCHIMERA|ECR_0|ECR_1|ECR_2|LATEST] prefix.gkpStore/  | perl -ane 'print join "\t", @F[0,-2,-1]; print "\n";' > prefix.?.clr
 CLR:    initial
 LATEST: final

MATED

 frg22mates.pl < prefix.frg > prerfix.mates

Subset

 touch file.uids
 gatekeeper -uid file.uids ...

Meryl

Get mer frequencies

 meryl -C -B -m 22 -s prefix.seq -o prefix.22mers

Dump mer sequences

 meryl -Dt -s prefix.22mers

Dump histogram

 meryl -Dh -s prefix.22mers | sort -nk2 -r | more

OBT

Overlap

Jobs

 merOverlapperSeedBatchSize     100000
 merOverlapperExtendBatchSize   75000
 
 overmerry.sh :       #frgs/merOverlapperSeedBatchSize   jobs -> seeds/ dir
 olap-from-seeds.sh : #frgs/merOverlapperExtendBatchSize jobs -> olaps/ dir

Dump

 overlapStore -d prefix.ovlStore > prefix.ovl.tab

 overlapStore -d prefix.ovlStore | awk '{print $1}' | uniq -c | awk '{print $2,$1}' > prefix.ovlStore.count.tmp
 cat prefix.ovlStore.count.tmp | perl -ane 'foreach ($p+1..$F[0]-1) { print "$_ 0\n" } $p=$F[0]; print $_'  > prefix.ovlStore.count
 rm prefix.ovlStore.count.tmp

Get stats

 overlapStats -G prefix.gkpStore -O prefix.ovlStore/ -o prefix

Unitiger

 tigStore -g prefix.gkpStore -t prefix.tigStore 2 -D properties | grep unitig_coverage_stat  > utg.properties

CGW

Consensus

 tigStore -g asm.gkpStore -t asm.tigStore/ 20 -D unitigs > unitigs.txt
 cat unitigs.txt | grep -v ^maID | perl -ane 'system "tigStore -g asm.gkpStore -t asm.tigStore 20 -d consensus -u  $F[0]\n";' >& unitigs.fasta.tmp
 cat unitigs.fasta.tmp  | grep -v null | sed 's/cns=//' | sed 's/-//g' | nl0 | tab2fasta.pl > unitigs.fasta
 rm unitigs.fasta.tmp

 tigStore -g asm.gkpStore -t asm.tigStore/ 20 -D contigs > contigs.txt
 cat contigs.txt | grep -v ^maID | perl -ane 'system "tigStore -g asm.gkpStore -t asm.tigStore 20 -d consensus -c  $F[0]\n";'>& contigs.fasta.tmp
 cat contigs.fasta.tmp | grep -v null | sed 's/cns=//' | sed 's/-//g' | nl0 | tab2fasta.pl > contigs.fasta
 rm contigs.fasta.tmp

Terminator

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 -i 1 > prefix.posmap.ctgfrgcnt
 cat prefix.posmap.frgscf | count.pl -i 1 > prefix.posmap.scffrgcnt
 cat prefix.posmap.ctgscf | count.pl -i 1 > prefix.posmap.scfctgcnt

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

 posmap2cvg.pl                                  < prefix.posmap.frgscf > prefix.posmap.scffrgcvg
 OLD/posmap2cvg.4.pl -mates prefix.posmap.mates < prefix.posmap.frgscf > prefix.posmap.scfmatecvg
 posmap2cvg.pl                                  < prefix.posmap.ctgscf > prefix.posmap.scfctgcvg

Get 0cvg regions

 posmap2cvg.pl -f prefix.posmap.scflen -Max 0 < prefix.posmap.frgscf  # scaff gaps
 posmap2cvg.pl -f prefix.posmap.ctglen -Max 0 < prefix.posmap.frgctg  # surrogates

Get links

 ~/bin/scf2scflnk.sh prefix                      
 ~/bin/ctg2ctglnk.sh prefix                      
 ...

Add ctg/scf into to the mates fils

 join12.pl prefix.posmap.mates prefix.posmap.frgctg > prefix.posmap.mates2.tmp
 join12.pl prefix.posmap.mates2.tmp prefix.posmap.frgscf > prefix.posmap.mates2
 rm prefix.posmap.mates2.tmp

Display graphs

cat prefix.posmap.scf2scflnk | ~/bin/tab2dot.pl | dot -Tpng -o prefix.posmap.scf2scflnk.png
cat prefix.posmap.ctg2ctglnk | ~/bin/tab2dot.pl | dot -Tpng -o prefix.posmap.ctg2ctglnk.png

Qc stats based on posmap files

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

Break scf/ctg pipeline

 ~/bin/breakPosmapKeep.amos

Get scaff file

 ~/bin/posmap2scaff.pl prefix.posmap.ctgscf

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";'

Qc

 caqc.pl -euid  prefix.asm

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

 wget ftp://ftp.ncbi.nih.gov/pub/TraceDB/porphyromonas_gingivalis_w83/anc.porphyromonas_gingivalis_w83.001.gz
 ...
 wget ftp://ftp.ncbi.nih.gov/pub/TraceDB/porphyromonas_gingivalis_w83/xml.porphyromonas_gingivalis_w83.001.gz

 /fs/sztmpscratch/dpuiu/porphyromonas_gingivalis_w83/

Complete genome at NCBI : NC_002950.2 ( 2,343,476bp 48.29%GC)

 bp_fetch.pl net::genbank:NC_002950.2 > porphyromonas_gingivalis_w83.1con

Input formatting

https://sourceforge.net/apps/mediawiki/wgs-assembler/index.php?title=Formatting_Inputs

Read counts

 zcat anc.*.gz | ~/bin/traceanc2anc.pl SEQ_LIB_ID INSERT_SIZE  INSERT_STDEV SVECTOR_CODE | count.pl

TA formatting

frg FORMAT2

TIs are frg uids

All libs

 tracedb-to-frg.pl -xml xml.porphyromonas_gingivalis_w83.001.gz
 tracedb-to-frg.pl -lib xml.porphyromonas_gingivalis_w83.001.gz
 tracedb-to-frg.pl -frg xml.porphyromonas_gingivalis_w83.001.gz
 =>
 porphyromonas_gingivalis_w83.1.lib.frg  
 porphyromonas_gingivalis_w83.2.001.frg.bz2  
 porphyromonas_gingivalis_w83.3.lkg.frg

One lib at a time

 tracedb-to-frg.pl -only T13146 -xml xml.porphyromonas_gingivalis_w83.001.gz >& ! tracedb-to-frg.log
 tracedb-to-frg.pl -only T13146 -lib xml.porphyromonas_gingivalis_w83.001.gz >>& tracedb-to-frg.log
 tracedb-to-frg.pl -only T13146 -frg xml.porphyromonas_gingivalis_w83.001.gz >>& tracedb-to-frg.log

454 SRA formatting

Illumina formatting

use fastqToCA (wgs-6.0)

 fastqToCA \
   -insertsize 3000 300 \
   -libraryname 1 \
   -type illumina \
   -fastq /fs/szattic-asmg4/Bees/Bombus_impatiens/s_1_1_sequence.txt,/fs/szattic-asmg4/Bees/Bombus_impatiens/s_1_2_sequence.txt | \
    sed 's/ori:I/ori:O/' \
   > 1.frg
 =>
 {LIB                                                                                                                                      
 act:A                                                                                                                                     
 acc:1                                                                                                                                     
 ori:O                                                                                                                                     
 mea:3000.000                                                                                                                              
 std:300.000                                                                                                                               
 src:                                                                                                                                      
 .                                                                                                                                         
 nft:10                                                                                                                                    
 fea:                                                                                                                                      
 forceBOGunitigger=1                                                                                                                       
 isNotRandom=0                                                                                                                             
 doNotTrustHomopolymerRuns=0                                                                                                               
 doRemoveDuplicateReads=0                                                                                                                  
 doNotQVTrim=0                                                                                                                             
 goodBadQVThreshold=0                                                                                                                      
 doNotOverlapTrim=0                                                                                                                        
 usePackedFragments=1                                                                                                                      
 illuminaFastQType=illumina                                                                                                                
 illuminaSequence=/fs/szattic-asmg4/Bees/Bombus_impatiens/s_1_1_sequence.txt,/fs/szattic-asmg4/Bees/Bombus_impatiens/s_1_2_sequence.txt    
 .                                                                                                                                         
 }

 gatekeeper \
   -o asm.gkpStore.BUILDING \
   -T -F  \
   -E asm.gkpStore.errorLog \
   1.frg 
 => Segmentation fault

FASTQ

This won't remove the linker:

 zcat SRR001351.fastq.gz | ~/bin/fastq2seq.pl  > SRR001351.seq
 zcat SRR001351.fastq.gz | ~/bin/fastq2qual.pl > SRR001351.qual
 convert-fasta-to-v2.pl -454 -l SRR001351 -s SRR001351.seq -q SRR001351.qual

SFF

 sffToCA -libraryname SRR001351 -output SRR001351.frg SRR001351.sff

Running

Command

 ~/wgs/Linux-amd64/bin/runCA -p pging -d testassembly porphyromonas_gingivalis_w83.* >& runCA.log

Dpuiu CA: Difference between revisions

Revision as of 03:11, 9 March 2010

Contents

Sourceforge

Run

Parameters

Gatekeeper

Build

Update

Delete

Dump

Meryl

OBT

Overlap

Unitiger

CGW

Consensus

Terminator

Log file & runtime (wgs-5.2)

Output processing

Posmap

Fasta

Asm

Qc

Scaffolds

AGP

Running subassemblies

Test datasets

Input formatting

Read counts

TA formatting

454 SRA formatting

Illumina formatting

FASTQ

SFF

Running

Navigation menu

Dpuiu CA: Difference between revisions

Revision as of 03:11, 9 March 2010

Sourceforge

Run

Parameters

Gatekeeper

Build

Update

Delete

Dump

Meryl

OBT

Overlap

Unitiger

CGW

Consensus

Terminator

Log file & runtime (wgs-5.2)

Output processing

Posmap

Fasta

Asm

Qc

Scaffolds

AGP

Running subassemblies

Test datasets

Input formatting

Read counts

TA formatting

454 SRA formatting

Illumina formatting

FASTQ

SFF

Running

Navigation menu

Search