Trim.Seqs is removing all my sequences

Commands in mothur
1

I have a data set and I am trying to filter and sort based on barcode. I have been trying to use the trim.seqs command to do this. I have an oligos file with the barcodes in it. I initially tried to run the command without a forward primer, and it put all the sequences in scrap with the error that they were removed due to the forward primer. I put in the initial sequence of the barcode which is the same for all barcodes as the forward primer and it still removed all the sequences with a forward primer error. I then tried to give it just the last base and allowed a pdiffs of 1. I then tried doing this with the trim.flow command and I still lost all the sequences. I don’t know what to do to fix the problem.

Using mother version 1.29.1

2

Could you post a sequence and the lines from the oligos file?

3

Sample Sequence:

ACACACACCTCAGACACACACACCAGGGGTTGGGTATGGGGAGGGGGTTCATAGTAGAAGAGCGACGGTGAGAGCTAAGGTCGGGGCGGTGATGTAGAGGGTGATGGTAGATGCTGAGACTGCCAAGGCACACAGGGGAT

oligos file:

forward C
#reverse G
barcode TCAGACACACAC A1
barcode TCAGTCTCTCTC A2
barcode TCAGACGCGCGC A3
barcode TCAGTGCGCGCG A4
barcode TCAGAGAGACAC A5
barcode TCAGATATATAT A6
barcode TCAGTGTGTATA A7
barcode TCAGTGTGTCTC A8
barcode TCAGTCTCTGTG A9
barcode TCAGTCTCATCA A10
barcode TCAGACACATAT A11
barcode TCAGTACATATA A12
barcode TCAGACAGTATA A13
barcode TCAGAGACTATA A14
barcode TCAGACGAGAGT A15
barcode TCAGTCACACTA A16
barcode TCAGTAGACACA A17
barcode TCAGTCTACTCA A18

4

Is there any solution? I’m facing the same problems and I don’t know what to do!

5

In the trim.seqs command, mothur looks for the linker, then barcode, then spacer, then forward primer. Since your barcode is not at the beginning of the sequence mothur can’t find it. If all the sequences have the same start, you could try adding a linker to the oligos file.

ACACACACCTCAGACACACACACCAGGGGTTGGGTATGGGGAGGGGGTTCATAGTAGAAGAGCGACGGTGA
GAGCTAAGGTCGGGGCGGTGATGTAGAGGGTGATGGTAGATGCTGAGACTGCCAAGGCACACAGGGGAT


oligos file:

linker ACACACACC
barcode TCAGACACACAC A1
barcode TCAGTCTCTCTC A2
…

6

Hello,

I seem to be suffering a similar problem. I have an oligos file set up similar to the example above.
However, I have looked through my sequences, and do not see a linker as was in the previous case.
I am thinking the problem occurs in the trim.flows step though as my trim flow file is empty?
I am following the 454 SOP.

Any suggestions?

Thanks!!