Hi,
I am a new user of mothur. I am working with a set of 8 samples. My starting materials are thus 16 different sff, 8 for bacterial sequences and 8 for Achaea.
I am having problems with the trim.seqs command. I get empty files.
I am following the SOP pipeline:
sffinfo
trim.flows
shhh.flows
After this step (performed separately for the 16 samples) I would like to merge my files into two files: one for bacteria and one for Achaea) to be able to make Beta diversity analysis between these samples.
merge.files(input=fileA-fileB….,output=AB…)
trim.seqs(fasta=.fasta, oligos= .oligos)
When I run trim.seqs, the output files generated are empty (except the scrap) and it is odd that the name do not contain a dot.
Output File Names:
ArchNestrim.fasta
ArchNesscrap.fasta
ArchNesgroup
It seems thus that everything is sent to the scrap.flows file.
I tried to reverse forward and reverse primers but I obtain the same results (ArchNestrim.fasta is blank).
Would you know how I could fix the problem?
Thank you for your help.
Nesrine LENCHI
What are the scrap codes in the .scrap.fasta file?
Thank you for your rapid answer,
The scrap codes are bf as far as I checked. Before I forget to say that I make sort of « positive control » with another dataset that works perfectly for a colleague (to be sure that is was not a problem with the barcodes of my sequences) and I got empty files for this dataset too. I re-installed the latest version of mothur but the results is the same.
Thank you again for your help
Nesrine LENCHI
It looks like you have the wrong barcodes and/or forward primer. Can you post part of your oligos file?
Here it is.
Forward CATGCTGCCTCCCGTAGGAGT
#reverse GAGTTTGATCNTGGCTCAG
Barcode AAAAAAAC TC2
Barcode AAAAAAAT TC5
Barcode AAAAAAAG Tctm_Arch
Barcode AAAAAACA ST_57
Barcode AAAAAACC ST_205
Barcode AAAAAACT B_RN
Barcode AAAAAACG TM
Barcode AAAAAATA ST_57_Arch
Barcode AAAAAATC ST_205_Arch
Barcode AAAAAATT B_RN_Arch
Barcode AAAAAATG TM_Arch
Barcode AAAAAAGA TC5_Arch
Barcode AAAAAAGC Tctm
Barcode AAAAAAGT GL
Barcode AAAAAAGG GL_Arch
Barcode AAAAACAA TC2_Arch
Thanks
Those aren’t real barcodes - if they are, get a new sequence provider. Tell Research and Testing (I assume that’s who this is from) to give you the correct barcodes and you should be in good shape. Someday they might explain why they do this other than to confuse users…