Problems after trim.seqs

I am very new to these analyses, I am following the 454 SOP protocol and I get an error message after this command:
> summary.seqs(fasta=filename.shhh.trim.fasta, name=filename.shhh.trim.names)
which followed the trim.seqs command

“trim.seqs(fasta=filename.shhh.fasta, name=filename.shhh.names, oligos=filename.oligos, pdiffs=2, bdiffs=1, maxhomop=8, minlength=200, flip=T, processors=2)”

The error message says:
[ERROR]: filename.shhh.trim.fasta is blank, aborting.
Using filename.fasta as input file for the fasta parameter.
[ERROR]: filename.shhh.trim.names is blank, aborting.

Using 2 processors.
[WARNING]: This command can take a namefile and you did not provide one. The current namefile is filename.shhh.trim.names which seems to match filename.fasta.

When I checked the files created after the trim.seqs command I noticed that the filename.shhh.trim.fasta and filename.shhh.trim.names files are zero bytes

Could you help me identify the problem?

Thank you

This actually looks like an issue with trim.flows scrapping all your sequences. Here’s a link you may find helpful, http://ω

Thank you,
That helped me, I found out that it was the order=B that resulted in those zero byte files.

I ran trim.seqs and my trim file result was 0 bytes. i don’t have my flow data so I can’t run trim.flow. Is the problem is with my oligos file?

thank you,

It could be. Have you looked at the scrap codes mothur is giving you?

k linker missing
b barcode missing
s spacer missing
f forward primer missing
r reverse primer missing
t too many differences to the linker, spacer, barcode, and forward primer
l too short or too long
h homopolymer length too long
n too many ambiguous bases

Also, if a barcode is missing, then you’ll also be missing the forward primer and the spacer if you’ve included one.


thank you, according to the scrap file I am missing the barccode and forward primer. how can I fix it (I attached below my oligos file)?

barcode TTGTTGCTGT Osnat1
barcode GTGTGGTTGT Osnat2
barcode TAGGTGGAAT Osnat3
barcode TGTAGGTGGA Osnat4
barcode TTAGTGGTGA Osnat5
barcode GTGAAGGTAA Osnat6
barcode TGTTGTGGTA Osnat7
barcode GTTGATGAGT Osnat8
barcode GGTCAGTGTA Osnat9
barcode GTAATGGAGT Osnat10
barcode CTCGTTATTC Osnat11
barcode GGAAGTAAGG Osnat12
barcode CGGTGTGTGT Osnat13
barcode CGTCTTCTTA Osnat14

Are you using the pdiffs and bdiffs parameters? Can you post a few sequences?