Died at MeGAMerge-1.1.pl line 280

[pdalcinmartins]

Hello,

It looks like Newbler run and then Minimus2 failed. Any suggestions? Could I start it again from Minimus2 maybe? Thanks!

[pdalcin@chimera megamerge]$ MeGAMerge-1.1.pl -cpu 15 megamerge_20171003 March2015_P7_Core1_Depth10-12cm_1092932.scaffolds.fa May2015_P7_Core1_10-12cm_1092938.scaffolds.fa September2015_P7_Core6_Depth10-12_1092944.scaffolds.fa March2015_P7_Core1_Depth1-3cm_1092931.scaffolds.fa May2015_P7_Core1_Depth1-3cm_1092937.scaffolds.fa September2015_P7_Core6_Depth1-3cm_1092943.scaffolds.fa March2015_P7_Core1_Depth19-21cm_1092933.scaffolds.fa May2015_P7_Core1_Depth19-21cm_1092939.scaffolds.fa September2015_P7_Core6_Depth19-21_1092945.scaffolds.fa March2015_P8_Core3_Depth10-12cm_1092935.scaffolds.fa May2015_P8_Core1_Depth10-12cm_1092941.scaffolds.fa September2015_P8_Core1_Depth10-12_1092947.scaffolds.fa March2015_P8_Core3_Depth1-3cm_1092934.scaffolds.fa May2015_P8_Core1_Depth1-3cm_1092940.scaffolds.fa September2015_P8_Core1_Depth1-3_1092946.scaffolds.fa March2015_P8_Core3_Depth19-21cm_1092936.scaffolds.fa May2015_P8_Core1_Depth19-21cm_1092942.scaffolds.fa Setember2015_P8_Core1_Depth19-21cm_1092948.scaffolds.fa
Running Newbler assembly with 406669127878 sequences
runAssembly -force -large -rip -mi 98 -ml 80 -pairt -cpu 15 -a 200 -o megamerge_20171003/newbler megamerge_20171003/newblerIn.fasta 1>/dev/null 2>/dev/null
Running Minimus2 with 25427709 sequences
minimus2 -D CONSERR=0.06 -D MINID=98 -D OVERLAP=80 megamerge_20171003/minimus.fasta 1>/dev/null 2>/dev/null
minimus2 -D CONSERR=0.06 -D MINID=98 -D OVERLAP=80 megamerge_20171003/minimus.fasta 1>/dev/null 2>/dev/null failed:
Died at /home2/opt/MeGAMerge-master/MeGAMerge-1.1.pl line 280.

[pdalcinmartins]

This is the minimus.fasta.runAmos.log file:

!!! 2017-10-04 00:16:47 Started by [email protected] on Wed Oct 4 00:16:47 2017

!!! 2017-10-04 00:16:47 Doing step 10: Building AMOS bank & Dumping reads
!!! 2017-10-04 00:16:47 Running: rm -fr megamerge_20171003/minimus.fasta.bnk
!!! 2017-10-04 00:16:47 Done! Elapsed time:0d 0h 0m 0s

!!! 2017-10-04 00:16:47 Doing step 11
!!! 2017-10-04 00:16:47 Running: /home2/opt/amos-master/bin/bank-transact -c -z -b megamerge_20171003/minimus.fasta.bnk -m megamerge_20171003/minimus.fasta.afg
START DATE: Wed Oct 4 00:16:47 2017
Bank is: megamerge_20171003/minimus.fasta.bnk
0% 100%
AFG ..................................................
Messages read: 50855420
Objects added: 50855420
Objects deleted: 0
Objects replaced: 0
END DATE: Wed Oct 4 00:32:19 2017
!!! 2017-10-04 00:32:20 Done! Elapsed time:0d 0h 15m 33s

!!! 2017-10-04 00:32:20 Doing step 12
!!! 2017-10-04 00:32:20 Running: /home2/opt/amos-master/bin/dumpreads megamerge_20171003/minimus.fasta.bnk -M 0 > megamerge_20171003/minimus.fasta.ref.seq
Objects seen: 25427709
Objects written: 25427709
!!! 2017-10-04 00:53:44 Done! Elapsed time:0d 0h 21m 24s

!!! 2017-10-04 00:53:44 Doing step 13
!!! 2017-10-04 00:53:44 Running: /home2/opt/amos-master/bin/dumpreads megamerge_20171003/minimus.fasta.bnk -m 0 > megamerge_20171003/minimus.fasta.qry.seq
Objects seen: 25427709
Objects written: 25427709
!!! 2017-10-04 01:15:32 Done! Elapsed time:0d 0h 21m 48s

!!! 2017-10-04 01:15:32 Doing step 20: Getting overlaps
!!! 2017-10-04 01:15:32 Running: : -maxmatch -c 80 megamerge_20171003/minimus.fasta.ref.seq megamerge_20171003/minimus.fasta.qry.seq -p megamerge_20171003/minimus.fasta
!!! 2017-10-04 01:15:32 Done! Elapsed time:0d 0h 0m 0s

!!! 2017-10-04 01:15:32 Doing step 21
!!! 2017-10-04 01:15:32 Running: /usr/local/bin/show-coords -H -c -l -o -r -I 98 megamerge_20171003/minimus.fasta.delta | /home2/opt/amos-master/bin/nucmerAnnotate | egrep 'BEGIN|END|CONTAIN|IDENTITY' > megamerge_20171003/minimus.fasta.coords
ERROR: Could not parse delta file, megamerge_20171003/minimus.fasta.delta
error no: 400
!!! 2017-10-04 01:15:32 Command: /usr/local/bin/show-coords -H -c -l -o -r -I 98 megamerge_20171003/minimus.fasta.delta | /home2/opt/amos-master/bin/nucmerAnnotate | egrep 'BEGIN|END|CONTAIN|IDENTITY' > megamerge_20171003/minimus.fasta.coords exited with status: 1
!!! END - Elapsed time: 0d 0h 58m 45s

[pdalcinmartins]

Ok, we spotted a problem. Missing info in the /home2/opt/amos-master/bin/minimus2 file below.

First problem: the nucmer pathway is not specified! (NUCMER=:) That is why it did not run and thus did not create the minimus.fasta.delta file.
The second one is that single dash before maxmatch in line 20:
20: $(NUCMER) -maxmatch -c $(OVERLAP) $(REFSEQ) $(QRYSEQ) -p $(PREFIX)
I know that here there is also a single dash, but when I used minimus2 -h at the terminal, that option was showed as double dashed.

so, I changed those two lines to be:
NUCMER=/usr/local/bin/nucmer
20: $(NUCMER) --maxmatch -c $(OVERLAP) $(REFSEQ) $(QRYSEQ) -p $(PREFIX)

We'll see how that goes...

---

#!/home2/opt/amos-master/bin/runAmos -C
#? minimus2 - The AMOS Pipeline for merging 2 assemblies

#? Usage:
#? minimus2 prefix
#? -D REFCOUNT= \ # Number of sequences in the 1st assembly ; (Required)
#? -D OVERLAP= \ # Assembly 1 vs 2 minimum overlap (Default 40bp)
#? -D CONSERR= \ # Maximum consensus error (0..1) (Default 0.06)
#? -D MINID= \ # Minimum overlap percent identity for alignments (Default 94)
#? -D MAXTRIM= # Maximum sequence trimming length (Default 20bp)

#--------------------------------------- USER DEFINED VALUES ------------------#

REFCOUNT= 0
MINID = 94
OVERLAP = 40
MAXTRIM = 20
WIGGLE = 15
CONSERR = 0.06

#------------------------------------------------------------------------------#

TGT = $(PREFIX).afg
BANK = $(PREFIX).bnk
REFSEQ = $(PREFIX).ref.seq
QRYSEQ = $(PREFIX).qry.seq
ALIGN = $(PREFIX).delta
COORDS = $(PREFIX).coords
OVLTAB = $(PREFIX).ovl
OVLAMOS = $(PREFIX).OVL
CONTIG = $(PREFIX).contig
FASTA = $(PREFIX).fasta

SINGLETONS = $(PREFIX).singletons
SINGLETONSEQ = $(PREFIX).singletons.seq

#------------------------------------------------------------------------------#

INPUTS = $(TGT) $(REFCOUNT)
OUTPUTS = $(CONTIG) $(FASTA)

#------------------------------------------------------------------------------#

BINDIR=/home2/opt/amos-master/bin
NUCMER=:
DELTAFILTER = /usr/local/bin/delta-filter
SHOWCOORDS = /usr/local/bin/show-coords

#------------------------------------------------------------------------------#

Building AMOS bank & Dumping reads

10: rm -fr $(BANK)
11: $(BINDIR)/bank-transact -c -z -b $(BANK) -m $(TGT)
12: $(BINDIR)/dumpreads $(BANK) -M $(REFCOUNT) > $(REFSEQ)
13: $(BINDIR)/dumpreads $(BANK) -m $(REFCOUNT) > $(QRYSEQ)

Getting overlaps

20: $(NUCMER) -maxmatch -c $(OVERLAP) $(REFSEQ) $(QRYSEQ) -p $(PREFIX)
21: $(SHOWCOORDS) -H -c -l -o -r -I $(MINID) $(ALIGN) | $(BINDIR)/nucmerAnnotate | egrep 'BEGIN|END|CONTAIN|IDENTITY' > $(COORDS)
22: $(BINDIR)/nucmer2ovl -ignore $(MAXTRIM) -tab $(COORDS) | $(BINDIR)/sort2 > $(OVLTAB)

Converting overlaps

23: $(BINDIR)/ovl2OVL $(OVLTAB) > $(OVLAMOS)

Loading overlaps to the bank

24: rm -f $(BANK)/OVL.*
25: $(BINDIR)/bank-transact -z -b $(BANK) -m $(OVLAMOS)

Running contigger

30: rm -f $(BANK)/LAY.*
31: $(BINDIR)/tigger -b $(BANK)

Running consensus

40: rm -f $(BANK)/CTG.*
41: $(BINDIR)/make-consensus -B -e $(CONSERR) -b $(BANK) -w $(WIGGLE)

Outputting contigs

50: $(BINDIR)/bank2contig $(BANK) > $(CONTIG)

Converting to FastA file

60: $(BINDIR)/bank2fasta -b $(BANK) > $(FASTA)

Getting singletons

70: $(BINDIR)/listReadPlacedStatus -S -E $(BANK) > $(SINGLETONS)
71: $(BINDIR)/dumpreads -e -E $(SINGLETONS) $(BANK) > $(SINGLETONSEQ)