make.contigs group file problem

Commands in mothur
1

Hello,

I am having trouble with my group file right at the beginning of the MiSeq SOP.
I am working with Illumina MiSeq V4 reads and I am using mothur v.1.36.1 on windows.

I made contigs using forward and reverse fastq files and when I attempted to screen sequences I got the following error message over and over again and about a third of my sequences are thrown out:
Your groupfile does not include the sequence please correct.

I then took a closer look at the group file (.contigs.groups) and it seems that some sequence names get cut off and as a result there is a shift and the following sequences won’t be recognized (at least that is what I am assuming)
here is my code:

mothur > make.contigs(file=test.files, processors=3)


Group count: V4_29 41611 V4_30 205103 V4_31 203345

Total of all groups is 450059


mothur > summary.seqs(fasta=test.trim.contigs.fasta, processors=3)

Using 3 processors.

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 296 296 0 3 1
2.5%-tile: 1 307 307 0 4 11252
25%-tile: 1 309 309 0 4 112515
Median: 1 310 310 0 4 225030
75%-tile: 1 310 310 1 5 337545
97.5%-tile: 1 317 317 17 7 438808
Maximum: 1 602 602 68 296 450059
Mean: 1 313.655 313.655 2.04722 4.53988

of Seqs: 450059



mothur > screen.seqs(fasta=test.trim.contigs.fasta, group=test.contigs.groups, summary=test.trim.contigs.summary, maxambig=0, minlength=307, maxlength=317)

Using 3 processors.
Your groupfile does not include the sequence M02973_22_000000000-AK66Y_1_2112_24715_6481 please correct.
Your groupfile does not include the sequence M02973_22_000000000-AK66Y_1_2112_25076_17339 please correct.
Your groupfile does not include the sequence M02973_22_000000000-AK66Y_1_2112_25077_13464 please correct.
Your groupfile does not include the sequence M02973_22_000000000-AK66Y_1_2112_25083_10665 please correct.
and so on…

mothur > summary.seqs(fasta=test.trim.contigs.good.fasta, processors=3)

Using 3 processors.

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 307 307 0 3 1
2.5%-tile: 1 308 308 0 4 7301
25%-tile: 1 309 309 0 4 73008
Median: 1 310 310 0 4 146015
75%-tile: 1 310 310 0 5 219022
97.5%-tile: 1 312 312 0 6 284729
Maximum: 1 317 317 0 12 292029
Mean: 1 309.921 309.921 0 4.47276

of Seqs: 292029

I appreaciate any hints you can give me!

Thanks,
Julia

2

Is your group file blank? Have you seen this post, make.contigs output - XXX.contigs.group blank

3

Hello,
thanks for your reply.
I am using fastq files and my group file was not blank, so that wasn’t the problem.
But I am happy to say that since my last post, I was able to get it working. I am not exactly sure what the actual problem was, but I think my path was maybe too long.
So after copying all my mothur files directly on the C: drive (C:\mothur) it seems to be working fine now.