Your count file does not include the sequence...

dlearman · September 11, 2013, 2:25pm

Hello,

I am in the process of analyzing some MiSeq 250PE data (16S, 515F/806R). I have been been through the SOP and I am now running one of my samples. At the mothur > screen.seqs step, I have gotten an error regarding missing sequences (pasted below). I am not sure how to deal with this error.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_24648_16337 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_25527_24646 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_26180_18930 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_26589_23793 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_27283_14101 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_27526_15744 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_28064_13998 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_4901_12572 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_5979_17408 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_7227_20645 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_7691_12299 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_7710_12305 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_7848_13677 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_8131_21493 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_8480_5582 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_9379_11699 please correct.

Any comments would be greatly appreciated.

thanks,
d

westcott · September 11, 2013, 5:35pm

Mismatches like this often occur when you forget to include a name or count file on a command where you used the associated fasta file. If you post the commands you ran so far, I may be able to spot it.

dlearman · September 11, 2013, 6:40pm

Here is my log file.

mothur > make.contigs(ffastq=16S_1_GCGATATATCGC_L001_R1_001.fastq, rfastq=16S_1_GCGATATATCGC_L001_R2_001.fastq)

It took 1712 secs to process 717180 sequences.

Output File Names:
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.fasta
16S_1_GCGATATATCGC_L001_R1_001.scrap.contigs.fasta
16S_1_GCGATATATCGC_L001_R1_001.contigs.report

mothur > summary.seqs(fasta=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.fasta)

Using 1 processors.

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 243 243 0 3 1
2.5%-tile: 1 252 252 0 3 17930
25%-tile: 1 253 253 0 4 179296
Median: 1 253 253 0 4 358591
75%-tile: 1 253 253 0 5 537886
97.5%-tile: 1 254 254 6 6 699251
Maximum: 1 501 500 97 250 717180
Mean: 1 254.219 254.219 0.562694 4.42737

of Seqs: 717180

Output File Names:
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.summary

mothur > screen.seqs(fasta=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.fasta, maxambig=0, maxlength=275)

Output File Names:
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.fasta
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.bad.accnos

It took 23 secs to screen 717180 sequences.

mothur > summary.seqs(fasta=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.fasta)

Using 1 processors.

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 244 244 0 3 1
2.5%-tile: 1 252 252 0 3 14807
25%-tile: 1 253 253 0 4 148062
Median: 1 253 253 0 4 296124
75%-tile: 1 253 253 0 5 444185
97.5%-tile: 1 254 254 0 6 577440
Maximum: 1 275 275 0 20 592246
Mean: 1 253.01 253.01 0 4.40333

of Seqs: 592246

Output File Names:
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.summary

mothur > get.current()

Current files saved by mothur:
fasta=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.fasta
summary=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.summary

mothur > unique.seqs(fasta=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.fasta)

Output File Names:
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.names
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.fasta

mothur > count.seqs(name=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.names)

Using 1 processors.
It took 2 secs to create a table for 592246 sequences.

Total number of sequences: 592246

Output File Names:
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.count_table

mothur > summary.seqs(count=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.count_table)

Using 16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.fasta as input file for the fasta parameter.

Using 1 processors.

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 244 244 0 3 1
2.5%-tile: 1 252 252 0 3 14807
25%-tile: 1 253 253 0 4 148062
Median: 1 253 253 0 4 296124
75%-tile: 1 253 253 0 5 444185
97.5%-tile: 1 254 254 0 6 577440
Maximum: 1 275 275 0 20 592246
Mean: 1 253.01 253.01 0 4.40333

of unique seqs: 173415

total # of seqs: 592246

Output File Names:
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.summary

mothur > pcr.seqs(fasta=silva.bacteria/silva.bacteria.fasta, start=11894, end=25319, keepdots=F, processors=2)
Output File Names:
silva.bacteria/silva.bacteria.pcr.fasta

It took 20 secs to screen 14956 sequences.

mothur > system(mv silva.bacteria/silva.bacteria.pcr.fasta silva.v4.fasta)

mothur > summary.seqs(fasta=silva.v4.fasta)

Using 2 processors.

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 13424 270 0 3 1
2.5%-tile: 1 13425 292 0 4 374
25%-tile: 1 13425 293 0 4 3740
Median: 1 13425 293 0 4 7479
75%-tile: 1 13425 293 0 5 11218
97.5%-tile: 1 13425 294 1 6 14583
Maximum: 3 13425 351 5 9 14956
Mean: 1.00074 13425 292.977 0.0573014 4.57014

of Seqs: 14956

Output File Names:
silva.v4.summary

mothur > align.seqs(fasta=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.fasta, reference=silva.v4.fasta)

Some of you sequences generated alignments that eliminated too many bases, a list is provided in 16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.flip.accnos. If you set the flip parameter to true mothur will try aligning the reverse compliment as well.
It took 369 secs to align 173415 sequences.

Output File Names:
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.align
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.align.report
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.flip.accnos

mothur > summary.seqs(fasta=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.align, count=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.count_table)

Using 2 processors.

Start End NBases Ambigs Polymer NumSeqs
Minimum: 0 0 0 0 1 1
2.5%-tile: 1968 11550 252 0 3 14807
25%-tile: 1968 11550 253 0 4 148062
Median: 1968 11550 253 0 4 296124
75%-tile: 1968 11550 253 0 5 444185
97.5%-tile: 1968 11550 254 0 6 577440
Maximum: 13425 13425 274 0 20 592246
Mean: 1970.02 11549.7 252.951 0 4.40253

of unique seqs: 173415

total # of seqs: 592246

Output File Names:
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.summary

mothur > screen.seqs(fasta=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.align, count=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.count_table, summary=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.

Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_24648_16337 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_25527_24646 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_26180_18930 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_26589_23793 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_27283_14101 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_27526_15744 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_28064_13998 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_4901_12572 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_5979_17408 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_7227_20645 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_7691_12299 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_7710_12305 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_7848_13677 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_8131_21493 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_8480_5582 please correct.
Your count file does not include the sequence HWI-M01141_82_A56CJ_1_2114_9379_11699 please correct.

Output File Names:
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.good.summary
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.good.align
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.bad.accnos
16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.good.count_table

It took 89 secs to screen 173415 sequences.

westcott · September 16, 2013, 10:48am

I think the problem may be here:

mothur > screen.seqs(fasta=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.align, count=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.count_table, summary=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.

I think you wanted to use summary=16S_1_GCGATATATCGC_L001_R1_001.trim.contigs.good.unique.summary.

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Your count file does not include the sequence...

of Seqs: 717180

of Seqs: 592246

of unique seqs: 173415

of Seqs: 14956

of unique seqs: 173415

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