Hi
I’m trying to follow the MiSeq SOP and got an error msg running summary.seqs after screen.seqs.
I am not sure I did things right. Could you please help me get over this so I can go forward?
I copy below the log file, with the error message at the end.
Thank you!
mothur >
make.file (inputdir=., type=fastq, prefix=stability)
Setting input directory to: C:\mothur\
[ERROR]: Invalid command.
[ERROR]: did not complete make.file .
mothur >
make.file (inputdir=., type=fastq, prefix=stability)
Setting input directory to: C:\mothur\
[ERROR]: Invalid command.
[ERROR]: did not complete make.file .
mothur >
make.file(inputdir=., type=fastq, prefix=stability)
Setting input directory to: C:\mothur\
Output File Names:
C:\mothur\stability.files
mothur >
make.contigs(file=stability.files, processors=4)
Using 4 processors.
>>>>> Processing file pair C:\mothur\13_S5_L001_R1_001.FASTQ - C:\mothur\13_S5_L001_R2_001.FASTQ (files 1 of 12) <<<<<
Making contigs...
Done.
It took 410 secs to assemble 464110 reads.
>>>>> Processing file pair C:\mothur\14_S35_L001_R1_001.FASTQ - C:\mothur\14_S35_L001_R2_001.FASTQ (files 2 of 12) <<<<<
Making contigs...
Done.
It took 209 secs to assemble 235161 reads.
>>>>> Processing file pair C:\mothur\15_S36_L001_R1_001.FASTQ - C:\mothur\15_S36_L001_R2_001.FASTQ (files 3 of 12) <<<<<
Making contigs...
Done.
It took 245 secs to assemble 273067 reads.
>>>>> Processing file pair C:\mothur\16_S6_L001_R1_001.FASTQ - C:\mothur\16_S6_L001_R2_001.FASTQ (files 4 of 12) <<<<<
Making contigs...
Done.
It took 295 secs to assemble 335804 reads.
>>>>> Processing file pair C:\mothur\3_S1_L001_R1_001.FASTQ - C:\mothur\3_S1_L001_R2_001.FASTQ (files 5 of 12) <<<<<
Making contigs...
Done.
It took 426 secs to assemble 472262 reads.
>>>>> Processing file pair C:\mothur\4_S2_L001_R1_001.FASTQ - C:\mothur\4_S2_L001_R2_001.FASTQ (files 6 of 12) <<<<<
Making contigs...
Done.
It took 328 secs to assemble 377911 reads.
>>>>> Processing file pair C:\mothur\5_S3_L001_R1_001.FASTQ - C:\mothur\5_S3_L001_R2_001.FASTQ (files 7 of 12) <<<<<
Making contigs...
Done.
It took 337 secs to assemble 382097 reads.
>>>>> Processing file pair C:\mothur\6_S33_L001_R1_001.FASTQ - C:\mothur\6_S33_L001_R2_001.FASTQ (files 8 of 12) <<<<<
Making contigs...
Done.
It took 283 secs to assemble 319146 reads.
>>>>> Processing file pair C:\mothur\7_S4_L001_R1_001.FASTQ - C:\mothur\7_S4_L001_R2_001.FASTQ (files 9 of 12) <<<<<
Making contigs...
Done.
It took 206 secs to assemble 232924 reads.
>>>>> Processing file pair C:\mothur\SP01_S102_L001_R1_001.FASTQ - C:\mothur\SP01_S102_L001_R2_001.FASTQ (files 10 of 12) <<<<<
Making contigs...
Done.
It took 88 secs to assemble 116068 reads.
>>>>> Processing file pair C:\mothur\SP02_S103_L001_R1_001.FASTQ - C:\mothur\SP02_S103_L001_R2_001.FASTQ (files 11 of 12) <<<<<
Making contigs...
Done.
It took 92 secs to assemble 120246 reads.
>>>>> Processing file pair C:\mothur\SP08_S104_L001_R1_001.FASTQ - C:\mothur\SP08_S104_L001_R2_001.FASTQ (files 12 of 12) <<<<<
Making contigs...
Done.
It took 85 secs to assemble 109512 reads.
Group count:
7A_11 116068
7A_12 377911
7B_11 120246
7B_12 382097
7C_11 472262
7C_12 319146
7E_12 232924
7G_12 109512
P2A_14 464110
P2B_14 235161
P2C_14 273067
P2D_14 335804
Total of all groups is 3438308
It took 3034 secs to process 3438308 sequences.
Output File Names:
C:\mothur\stability.trim.contigs.fasta
C:\mothur\stability.trim.contigs.qual
C:\mothur\stability.scrap.contigs.fasta
C:\mothur\stability.scrap.contigs.qual
C:\mothur\stability.contigs.report
C:\mothur\stability.contigs.groups
mothur >
summary.seqs(fasta=stability.trim.contigs.fasta)
Using 4 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 35 35 0 3 1
2.5%-tile: 1 439 439 0 4 85958
25%-tile: 1 440 440 0 4 859578
Median: 1 445 445 1 5 1719155
75%-tile: 1 464 464 6 6 2578732
97.5%-tile: 1 469 469 23 7 3352351
Maximum: 1 602 602 260 300 3438308
Mean: 1 451 451 4 5
# of Seqs: 3438308
It took 92 secs to summarize 3438308 sequences.
Output File Names:
C:\mothur\stability.trim.contigs.summary
mothur >
screen.seqs(fasta=stability.trim.contigs.fasta, group=stability.contigs.groups, summary=stability.trim.contigs.summary, maxambig=0, minlength=439, maxlength=469, qfile=stability.trim.contigs.qual)
Using 4 processors.
It took 58 secs to screen 3438308 sequences, removed 2250012.
/******************************************/
Running command: remove.seqs(accnos=stability.trim.contigs.bad.accnos.temp, group=stability.contigs.groups, qfile=stability.trim.contigs.qual)
Removed 2250012 sequences from your group file.
Removed 2250012 sequences from your quality file.
Output File Names:
stability.contigs.pick.groups
stability.trim.contigs.pick.qual
/******************************************/
Output File Names:
stability.trim.contigs.good.summary
stability.trim.contigs.good.fasta
stability.trim.contigs.bad.accnos
stability.contigs.good.groups
stability.trim.contigs.good.qual
It took 247 secs to screen 3438308 sequences.
mothur >
summary.seqs(count=stability.trim.contigs.good.count_table)
Using stability.trim.contigs.good.unique.fasta as input file for the fasta parameter.
Using 4 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 439 439 0 3 1
2.5%-tile: 1 440 440 0 4 29708
25%-tile: 1 441 441 0 4 297075
Median: 1 445 445 0 5 594149
75%-tile: 1 465 465 0 6 891223
97.5%-tile: 1 469 469 0 6 1158589
Maximum: 1 469 469 0 177 1188296
Mean: 1 451 451 0 5
# of unique seqs: 786570
total # of seqs: 1188296
It took 22 secs to summarize 1188296 sequences.
Output File Names:
stability.trim.contigs.good.unique.summary
mothur >
align.seqs(fasta=ecoliv3v4.fasta, reference=silva.seed_v132.align)
Using 4 processors.
Reading in the silva.seed_v132.align template sequences... DONE.
It took 19 to read 11180 sequences.
Aligning sequences from ecoliv3v4.fasta ...
Reducing processors to 1.
It took 0 secs to align 1 sequences.
It took 0 seconds to align 1 sequences.
Output File Names:
ecoliv3v4.align
ecoliv3v4.align.report
mothur >
summary.seqs(fasta=ecoliv3v4.align)
Using 4 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 6388 25316 465 0 6 1
2.5%-tile: 6388 25316 465 0 6 1
25%-tile: 6388 25316 465 0 6 1
Median: 6388 25316 465 0 6 1
75%-tile: 6388 25316 465 0 6 1
97.5%-tile: 6388 25316 465 0 6 1
Maximum: 6388 25316 465 0 6 1
Mean: 6388 25316 465 0 6
# of Seqs: 1
It took 0 secs to summarize 1 sequences.
Output File Names:
ecoliv3v4.summary
mothur >
pcr.seqs(fasta=silva.seed_v132.align, start=6388, end=25316, keepdots=F, processors=8)
Using 8 processors.
[NOTE]: no sequences were bad, removing silva.seed_v132.bad.accnos
It took 17 secs to screen 11180 sequences.
Output File Names:
silva.seed_v132.pcr.align
mothur >
rename.file(input=silva.seed_v132.pcr.align, new=silva.v3v4.fasta)
Current files saved by mothur:
fasta=silva.seed_v132.pcr.align
processors=8
summary=ecoliv3v4.summary
mothur >
summary.seqs(fasta=silva.v3v4.fasta)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 17056 415 0 3 1
2.5%-tile: 1 18928 438 0 4 280
25%-tile: 1 18928 444 0 4 2796
Median: 1 18928 464 0 5 5591
75%-tile: 1 18928 465 0 6 8386
97.5%-tile: 1 18928 607 1 6 10901
Maximum: 3 18928 1274 5 12 11180
Mean: 1 18927 488 0 5
# of Seqs: 11180
It took 5 secs to summarize 11180 sequences.
Output File Names:
silva.v3v4.summary
mothur >
align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.v3v4.fasta)
Using 8 processors.
Reading in the silva.v3v4.fasta template sequences... DONE.
It took 8 to read 11180 sequences.
Aligning sequences from stability.trim.contigs.good.unique.fasta ...
It took 1493 secs to align 786570 sequences.
[WARNING]: 128 of your sequences generated alignments that eliminated too many bases, a list is provided in stability.trim.contigs.good.unique.flip.accnos.
[NOTE]: 119 of your sequences were reversed to produce a better alignment.
It took 1498 seconds to align 786570 sequences.
Output File Names:
stability.trim.contigs.good.unique.align
stability.trim.contigs.good.unique.align.report
stability.trim.contigs.good.unique.flip.accnos
mothur >
summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 17 3 0 1 1
2.5%-tile: 1 18928 439 0 4 29708
25%-tile: 1 18928 439 0 4 297075
Median: 1 18928 444 0 5 594149
75%-tile: 1 18928 464 0 6 891223
97.5%-tile: 1 18928 465 0 6 1158589
Maximum: 18910 18928 468 0 175 1188296
Mean: 1 18926 449 0 5
# of unique seqs: 786570
total # of seqs: 1188296
It took 516 secs to summarize 1188296 sequences.
Output File Names:
stability.trim.contigs.good.unique.summary
mothur >
screen.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table, summary=stability.trim.contigs.good.unique.summary, start=1, end=18928, maxhomop=8)
Using 8 processors.
It took 419 secs to screen 786570 sequences, removed 8994.
/******************************************/
Running command: remove.seqs(accnos=stability.trim.contigs.good.unique.bad.accnos.temp, count=stability.trim.contigs.good.count_table)
Removed 9052 sequences from your count file.
Output File Names:
stability.trim.contigs.good.pick.count_table
/******************************************/
Output File Names:
stability.trim.contigs.good.unique.good.summary
stability.trim.contigs.good.unique.good.align
stability.trim.contigs.good.unique.bad.accnos
stability.trim.contigs.good.good.count_table
It took 652 secs to screen 786570 sequences.
mothur >
summary.seqs(fasta=current, count=current)
Using stability.trim.contigs.good.good.count_table as input file for the count parameter.
Using stability.trim.contigs.good.unique.good.align as input file for the fasta parameter.
Using 8 processors.
[ERROR]: Your count file contains 777576 unique sequences, but your fasta file contains 604417. File mismatch detected, quitting command.
mothur >
quit