Precluster.fasta has zero sequences

1

Hi!
I am running mothur v.1.48.0 for a single sample.
The precluster.fasta file is empty although it is showing the following:
[WARNING]: /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.good.unique.good.filter.unique.fasta does not contain any sequence from the .accnos file.

Selected 0 sequences from /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.good.unique.good.filter.unique.fasta.

Output File Names:

/Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.good.unique.good.filter.unique.precluster.fasta

/******************************************/

Done.

It took 137 secs to cluster 92689 sequences.

Using 4 processors.

Output File Names:

/Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.good.unique.good.filter.unique.precluster.fasta

/Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.good.unique.good.filter.unique.precluster.count_table

/Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.good.unique.good.filter.unique.precluster.map

Kindly suggest to correct this error

2

I have used the following commands:
make.contigs(ffastq=KSP_PE_R1.fastq, rfastq=KSP_PE_R2.fastq)
summary.seqs(fasta=current, count=current)
screen.seqs(fasta=current, count=current, maxambig=0, maxlength=500, maxhomop=8)
summary.seqs(fasta=current, count=current)
screen.seqs(fasta=current,count=current, maxambig=0, minlength=300, maxhomop=8)
unique.seqs(fasta=current,count=current)
align.seqs(fasta=current, reference=/Volumes/Anusha/mothur/silva.nr_v38_1/silva.nr_v138_1.align)
summary.seqs(fasta=current, count=current)
screen.seqs(fasta=current, count=current, start=6388, end=25316)
filter.seqs(fasta=current, vertical=T, trump=.)
unique.seqs(fasta=current, count=current)
pre.cluster(fasta=current, count=current)
Total number of sequences before precluster was 92689.

pre.cluster removed 2335 sequences.

/******************************************/

[WARNING]: /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.good.unique.good.filter.unique.fasta does not contain any sequence from the .accnos file.

Selected 0 sequences from /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.good.unique.good.filter.unique.fasta.

Output File Names:

/Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.good.unique.good.filter.unique.precluster.fasta

/******************************************/

Done.

It took 137 secs to cluster 92689 sequences.

3

Can you try to rerun the unique.seqs and pre.cluster steps? Iā€™d be curious to see what the output of summary.seqs is after running unique.seqs when you include the count file

Pat

4

Thank you for attending to this.
As per your suggestion, I reran the unique.seqs and precluster.seq. For unique.seqs, here is the summary:
mothur > summary.seqs(fasta=current, count=current)

Using /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.unique.good.filter.count_table as input file for the count parameter.

Using /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.unique.good.filter.unique.fasta as input file for the fasta parameter.

Using 4 processors.

Start End NBases Ambigs Polymer NumSeqs

Minimum: 1 1439 402 0 3 1

2.5%-tile: 1 1440 440 0 4 2407

25%-tile: 1 1440 441 0 5 24067

Median: 1 1440 462 0 6 48133

75%-tile: 1 1440 465 0 6 72199

97.5%-tile: 1 1440 466 0 6 93858

Maximum: 2 1440 498 0 8 96264

Mean: 1 1439 455 0 5

of unique seqs: 92689

total # of seqs: 96264

It took 6 secs to summarize 96264 sequences.

Output File Names:

/Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.unique.good.filter.unique.summary

While for precluster.seqs, the summary is as follows:

summary.seqs(fasta=current, count=current)

Using /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.unique.good.filter.unique.precluster.count_table as input file for the count parameter.

Using /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.unique.good.filter.unique.precluster.fasta as input file for the fasta parameter.

[ERROR]: /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.unique.good.filter.unique.precluster.fasta is blank, aborting.

Using /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.unique.good.filter.unique.precluster.fasta as input file for the fasta parameter.

Using 4 processors.

[ERROR]: /Volumes/Anusha/mothur/CoE1/KSP_PE_R1.trim.contigs.good.unique.good.filter.unique.precluster.fasta is blank. Please correct.

Error in reading your fastafile, at position -1. Blank name.

1 Like
5

Could you send KSP_PE_R1.trim.contigs.good.unique.good.filter.count_table and KSP_PE_R1.trim.contigs.good.unique.good.filter.unique.fasta to mothur.bugs@gmail.com so I can take a closer look?

6

I have forwarded the files, could you kindly check it.
Thank you