Failure during Minimus2 Step

[nfellaby]

So I think I may have found the root of the error but I am unsure how to resolve it.

Reads are from HiSeq 2500, 101 bp paired end reads

Here is the output from the debug mode, with force write (-force) enabled:

~/bin/MeGAMerge/MeGAMerge-1.1.pl -force -d -cpu=32 DF1_MegaMerge/ 1-STH063DF1_CGTACTAG-TAGATCGC_L003_R1_001.fastq 1-STH063DF1_CGTACTAG-TAGATCGC_L003_R2_001.fastq
COMMAND
perl /pub40/nfellaby/bin/MeGAMerge/MeGAMerge-1.1.pl -force -d -cpu=32 DF1_MegaMerge/ 1-STH063DF1_CGTACTAG-TAGATCGC_L003_R1_001.fastq 1-STH063DF1_CGTACTAG-TAGATCGC_L003_R2_001.fastq
The Merged FASTA will be stored in DF1_MegaMerge//MergedContigs.fasta
Reading 1-STH063DF1_CGTACTAG-TAGATCGC_L003_R1_001.fastq
Reading 1-STH063DF1_CGTACTAG-TAGATCGC_L003_R2_001.fastq
Running Newbler assembly with 4991773 sequences
runAssembly -force -large -rip -mi 98 -ml 80 -pairt -cpu 32 -a 200 -o DF1_MegaMerge//newbler DF1_MegaMerge//newblerIn.fasta
Initialized assembly project directory DF1_MegaMerge//newbler
1 read file successfully added.
newblerIn.fasta (Fasta dataset)
Assembly computation starting at: Tue Jul 26 13:19:23 2016 (v2.9 (20130529_1641))
Indexing newblerIn.fasta...
Warning: No quality scores file found.
-> 3310606 reads, 2193389201 bases.
Warning: Suspected 5' primer CGTACTAGTAGATCGC, 438341 exact matches found.
Warning: Suspected 3' primer CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC, 9273 exact matches found (displayed as the reverse complement in reads).
Setting up overlap detection...
-> 457701 of 457701, 457701 reads to align
Starting seed building...
-> 457701 of 457701
Building a tree for 4913601 seeds...
Computing alignments...
-> 457000 of 457701
7690 sequences marked as repeats, 7689 in alignments, removing from 2433 chords...
-> 457701 of 457701
Checkpointing...
Detangling alignments...
-> Level 4, Phase 9, Round 2...
Checkpointing...
Building contigs/scaffolds...
-> 32 large contigs, 276 all contigs
Computing signals...
-> 168066 of 168066...
Generating output...
-> 168066 of 168066...
Assembly computation succeeded at: Tue Jul 26 13:33:20 2016
423325 newbler singletons sequences
Checking 454PairAlign.txt
Unique Newbler Singletons (>=200 bp): 174250
opening DF1_MegaMerge//minimus.fasta for writing
opening DF1_MegaMerge//largefile.fasta for reading
opening DF1_MegaMerge//newbler/All.fasta for reading
Running Minimus2 with 1539831 sequences
Error: Temporary read file /tmp/tmp.18421.seq was not formatted as expected at line 8033:
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
toAmos -s DF1_MegaMerge//minimus.fasta -o DF1_MegaMerge//minimus.fasta.afg failed:
Died at /pub40/nfellaby/bin/MeGAMerge/MeGAMerge-1.1.pl line 275.

What I would say about the sequence file is that it contains huge chunks of 'N' inserts:
e.g.
#1

@HISEQ:99:C5LAVANXX:3:1101:2978:1923 1:N:0:CGTACTAGTAGATCGCA
GTGCAATGCTGTGATCTCGGCTAACCACAACCTCCGCCTCCCAGGTTCAAGCGATTCTCC
TGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGCATGCGCCACCACACCTGGCTAATTTT
GTA+=<<@FGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGDGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
BGGGGGGG@HISEQ:99:C5LAVANXX:3:1101:3136:1908 1:N:0:CGTACTAGT
AGATCGCAATGAAGTAGTAAAACCTGCATCAAGGTTTATGGTTTCATTCGAAAAACTGCC
TCCAAAAGAAAATTATTTTTAAAAAGAAGATTTAGAAAAAATTACCTAATTTCCATTTTA
AATGACCCTCT+=<@BGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGEGGGGGGG@HISEQ:99:C5LAVANXX:3:1101:3083:1926 1:N:0:C
GTACTAGTAGATCGCCTGAGGATCTCTAAGTATTTAACATCAAAATACTGAAAATTGCCA
TTTTTCACCATTAATTGTAATTCAAATGGCATTTGATTAATGGATGTTCACCTTTTTCTA
AAAATTAAAAAATAAGATT+=<@BGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGG@HISEQ:99:C5LAVANXX:3:1101:3486:1940
1:N:0:CGTACTAGTAGATCGCTTCTAGCTACAAGAGACACAGTGGTCAGCAAAAGAAA
CACTGCCAGCCTTCCCAGAGCTTATAGATTAGTGAGGGAGAGGGTGAATAACCAGATAAA
CAAACGCTCGTCAGTGCTATGAAAGAA+<<@BGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@HISEQ:99:C5LAVANXX:3:1101:3
425:1958 1:N:0:CGTACTAGTAGATCGCCACTATGCTTGGCAGCGTGGTGTGGATAA
TAACTGGGTGCCCTGGGAGGGGTGAGGACACTCCAAGCTACAGAAGATCAGGGAAGAGGG
AGATCAAAGTGGCCTAGAGATTTTAAGAAAATCTT+=<@BGGFGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGEGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@HISEQ:99:C5LAVANXX:
3:1101:3282:1985 1:N:0:CGTACTAGTAGATCGCATCCCCACGTCATGGGACTCA
GTTAAAGGTAATTGAATCATGGGGGTGATTTCCCCCATGCTATTCTCATGATAGTGAGTA
ACTTCTTATGAGATCCGATGGTTTTATAAGGGGTTTCTTCCTT+=<@bgggggggggggg
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@HISEQ:99:C5
LAVANXX:3:1101:3700:1927 1:N:0:CGTACTAGTAGATCGCTGCCAAATAGTTC
TCATAAATGTTACTTTCAAGTTTATGTAAACAATATTATGAAGGGCTTTGATTCATCACA
AAATTTAAAACTTTTTAAAATAACATTTCCCAAGTACATTTAAAGTAAGAC+=@bfgggg
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@HISE
Q:99:C5LAVANXX:3:1101:3538:1931 1:N:0:CGTACTAGTAGATCGCATAAAA
CTGAGCCTGCATGGGAATTTCAACATTCCAAAATAACACAAACAGCATTATTGGACCCTT
TGGGTCAAATAAATCATTTACCAAGAGAAGAAGGAAAGGAATTGATGGGGATAGGAAT+=
<BBGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGG@HISEQ:99:C5LAVANXX:3:1101:3695:1947 1:N:0:CGTACTAGTAGATC
GCTGGAGCAGTTTCGAAACACACTATTTGTAGAATGTGCAAGTGGATATTTAGGCCTCTC
TGAGGATTTCGTTGGAAACGGGATAAACCGCACAGAACTAAACAGAAGCATTCTCAGAAC
CTTCTT+<<@FGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCEGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGEGGGGGGGGGGFDFGGGGGGGEGGGGGGGGGGGGGGGGFGGG
GGGGGGGGFF@HISEQ:99:C5LAVANXX:3:1101:3778:1935 1:N:0:CGTACTA
GAAGATCGCCATAAAGTATCTCATCCTCAACTCACCCTCCATGGGTGATGGCTCTTGTCT
GTGGGGTTTACGGGAATTGGGTGTGTCCCTGGAAAAGGCTTCCCCAGGTGGTGGTGGTTG
TGGGGATTAAAG+<BGCFGGGGGGGCG

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Whilst I am unsure about how the program is stitching these sections together, or whether they are placeholders, I am pretty sure that these are causing the issue..

Any suggestions would be marvellous:
Machine Stats:
Constant Metrics
CPU Count 64 CPUs
CPU Speed 3300 MHz
Memory Total 529195296 KB
Swap Space Total 0 KB

x86_64 GNU/Linux
Kernel: 3.16.7-ckt25

Thanks for your time,
nicholas.

[nfellaby]

to add, the sequence in question which appears to be incorrectly formatted is actually:

#72
@HISEQ:99:C5LAVANXX:3:1101:18438:3091 1:N:0:CGTACTAGTAGATCGC
GGACTACTGACTAACAAATTCCCCCAGTTAAAATATGGCTTAAAATTCCAGGCCAGCCAC
AGTGACTCATGCCTGTGATCCCAGCACTTTGGGAGGCTGAAGTGGGAGGATTACTCGAGC
TCAGG+BBCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGEGGGGGGGGGG
GGGGGGGGGG@HISEQ:99:C5LAVANXX:3:1101:18336:3229 1:N:0:CGTACT
AGTAGATCGCCTCAAGTACCCAGGGACACAAAAACTGCGGAAGGCCGCAGGGACCTCTGC
CTAGGAAAGCCAGGTATTGTCCAAGGTTTCTCCCCATGTGATAGTCTAAAATATGGCCTC
GTGGGAAGGGAAAGA+ABBBBGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGG@HISEQ:99:C5LAVANXX:3:1101:18566:3017 1
:N:0:CGTACTAGTAGATCGCTCTAAATACCATCTAAATTGAATGTCTGTGAAACTCAAG
TTGGTACCCATCTGGAGGCAAATTCCCTTCTAACTCTGAGTCTGTAAAATAAAACAAGTG
ATGTGTTTCCAAAATACAATGGAGGG+CBCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@HISEQ:99:C5LAVANXX:3:1101:1
8625:3248 1:N:0:CGTACTAGTAGATCGCGTCTACAGCAAATTTGTTCTTCAGTGTT
TCTAGTATCAGTTTTTGATCAATCATTAATCAAAGTTGCAATAAAAAGATAATCTTCTCA
GGACTAGGCTATAAAGGTCCTGGCTGCAACCTTAAAA+BBBBGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGEGGGG
GGGGGGGGGGGGGGFGGGGGGGGGGGGCGGGGG@GGGGGGGG@HISEQ:99:C5LAVANX
X:3:1101:18999:3048 1:N:0:CGTACTAGTAGATCGCCTTTACTTTCTTACTCAG
TCCTTTTATCATCGGAATTAGAGCCTTCTAGCCAGTTATTTTTCTCAGAGGATTTGTTTT
TCGACCTGGCATCTCACTTCAGGACTTTGTATCTGTGGAAATATGGT+BBBBBFGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGEG@HISEQ:
99:C5LAVANXX:3:1101:18830:3234 1:N:0:CGTACTAGTAGATCGCCTTGTTT
CACTGCAACCTCCGCCTTCTGGGTTCAAGTGATTCTCTTGCCTCAGCCTCCCAAGTAGCT
GAGACTACAGGTGCTTGTCACCACGCCTGGCTAATTTTTTTTTTTTTTTTTTTTTGTT+B
BCBGGGGGGGGGGGGGGGGGGFDEGGGGGGGGGGGGGGFGGGGGEGGGGGGGGGGGGGGG
GGGGGGFGGGGGGGGFGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG##
###@HISEQ:99:C5LAVANXX:3:1101:18799:3241 1:N:0:CGTACTAGTAGAT
CGCAATGGAATCAACACGAGTGGAATGGCATGGATTGGAATGGAATGGAATGGAATCAAC
CCGAGTACAGGGGAATGGAATGGAATGGAATGCAATGGAATGGAATCATCCGTAATGGAA
TGGAAAG+:3@

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[scholzmb]

MeGAmerge is not intended to be fed raw fastq files. You will first need to assemble the data using a current tool (MetaSpades, eg, using multiple assembly parameters. MeGAMerge is a post assembly tool to improve assemblies by leveraging the fact that no two assemblers seem to assemble the same data into the same contigs.