filter.seqs error PLEASE HELP!

Commands in mothur
1

Hi,

I am following the MiSeq SOP, and I have an error I didn’t find an answer to in the forum:

mothur > filter.seqs(fasta=theonella.trim.contigs.good.unique.pick.good.align, vertical=T, trump=.)


Unable to open 30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.good.align. Trying default /data/apps/mothur/1.37.4/theonella.trim.contigs.good.unique.pick.good.align Unable to open /data/apps/mothur/1.37.4/theonella.trim.contigs.good.unique.pick.good.align. Trying output directory /data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.good.align

Using 20 processors.
Error in reading your fastafile, at position -1. Blank name.
Creating Filter…

I am using mothur version 1.37.4. It is a linux OS (slurm)

Thanks

Maya

2

PS:

These are the commands I used prior the filter.seq:

mothur > pcr.seqs(fasta=/data/home/steindler/PublicStorage/Silva/Silva_123/silva.nr_v123.align, start=11894, end=25319, keepdots=F, processors=20)

mothur > summary.seqs(fasta=silva.nr_v123.pcr.align)

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 9876 212 0 3 1
2.5%-tile: 1 13425 292 0 4 4311
25%-tile: 1 13425 293 0 4 43105
Median: 1 13425 293 0 5 86210
75%-tile: 1 13425 293 0 5 129314
97.5%-tile: 1 13425 459 1 6 168108
Maximum: 4225 13425 1122 5 16 172418
Mean: 1.14112 13424.9 308.769 0.0498904 4.74949

of Seqs: 172418


mothur > align.seqs(fasta=theonella.trim.contigs.good.unique.pick.fasta, reference=silva.nr_v123.pcr.align, processors=20, flip=t)

…
Some of your sequences generated alignments that eliminated too many bases, a list is provided in /data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.flip.accnos. If the reverse compliment proved to be better it was reported.
It took 1211 secs to align 663933 sequences.


Output File Names: /data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.align /data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.align.report /data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.flip.accnos

mothur > summary.seqs(fasta=current, count=current, processors=20)


Start End NBases Ambigs Polymer NumSeqs Minimum: 1 1231 2 0 1 1 2.5%-tile: 1 13425 292 0 4 16599 25%-tile: 1 13425 293 0 4 165984 Median: 1 13425 293 0 5 331967 75%-tile: 1 13425 293 0 5 497950 97.5%-tile: 1 13425 294 0 8 647335 Maximum: 13424 13425 308 0 19 663933 Mean: 1.11931 13424.9 293.055 0 4.85941 # of Seqs: 663933

Output File Names:
/data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.summary

It took 30 secs to summarize 663933 sequences.

mothur > screen.seqs(fasta=theonella.trim.contigs.good.unique.pick.align, count=theonella.trim.contigs.good.pick.count_table, summary=theonella.trim.contigs.good.unique.pick.summary, optimize=start, end=13425, maxhomop=8)

Output File Names:
/data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.good.summary
/data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.good.align
/data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.bad.accnos
/data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.pick.good.count_table


mothur > summary.seqs(fasta=current, count=current, processors=20)

Start End NBases Ambigs Polymer NumSeqs Minimum: 1 13425 290 0 3 1 2.5%-tile: 1 13425 292 0 4 17022 25%-tile: 1 13425 293 0 4 170212 Median: 1 13425 293 0 5 340424 75%-tile: 1 13425 293 0 5 510636 75%-tile: 1 13425 293 0 5 510636 97.5%-tile: 1 13425 294 0 8 663826 Maximum: 1 13425 301 0 8 680847 Mean: 0.999966 13424.5 293.046 0 4.85449 # of unique seqs: 663122 total # of seqs: 680847

Output File Names:
/data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.good.summary


mothur > filter.seqs(fasta=theonella.trim.contigs.good.unique.pick.good.align, vertical=T, trump=.)
Unable to open 30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.good.align. Trying default /data/apps/mothur/1.37.4/theonella.trim.contigs.good.unique.pick.good.align Unable to open /data/apps/mothur/1.37.4/theonella.trim.contigs.good.unique.pick.good.align. Trying output directory /data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.good.align

Using 20 processors.
Error in reading your fastafile, at position -1. Blank name.
Creating Filter…

3

The filter.seqs command allows for processing multiple files at a time separated by dashes. Since your full pathname contains a dash mothur is trying to open “30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.good.align” instead of “/data/home/steindler/mbritstei/petrosia_thonella_16S/MiSeq140_Maya_12437-30241234/fastq/theonella/theonella.trim.contigs.good.unique.pick.good.align” thinking you provided two files. Can you try removing the “-” from the full path name? Or putting the file in a location without a dash in the full path?

4

I can, and it worked! thank you so much!!!