sampleName in oligos file

Commands in mothur
1

Hi! Simple question:
I have a list of sff files from the sequencing facility, one for each sample. My understanding is that I want to use sff.multiple to start things off (and not merge.sfffiles, which I tried first), but I’m not sure how to set up the oligos file.
I have all the sff file names in a .files file, which merge.sfffiles read fine, but are these sff file names equivalent to the sample names for setting up the oligos file? Or is it the SampleID that the sequencing facility used (based on plate position)?
The sequencing facility sent me a “LinkerPrimerSequence,” which is the same for all samples and I assume is my “forward” primer if I’m setting it up as in the oligos from the Schloss_SOP, and then I have a barcode for each sample (but, again, unsure how to get mother to read the sampleName correctly)
Many thanks,
Beth

2

For your oligos file you want something like this:

GQY1XT001.oligos:

forward CCGTCAATTCMTTTRAGT
barcode AATGGTAC F003D000
barcode AACCTGGC F003D002
barcode TTCGTGGC F003D004
barcode TTCTTGAC F003D006
barcode TTCGCGAC F003D008
barcode TCCAGAAC F003D142
barcode AAGGCCTC F003D144
barcode TGACCGTC F003D146
barcode AGGTTGTC F003D148
barcode TGGTGAAC F003D150
barcode AACCGTGTC MOCK.GQY1XT001

For your file file:

sffFiles.txt:

GQY1XT001.F003D000.sff GQY1XT001.oligos
GQY1XT001.F003D002.sff GQY1XT001.oligos
GQY1XT001.F003D004.sff GQY1XT001.oligos
GQY1XT001.F003D006.sff GQY1XT001.oligos
GQY1XT001.F003D008.sff GQY1XT001.oligos
GQY1XT001.F003D142.sff GQY1XT001.oligos
GQY1XT001.F003D144.sff GQY1XT001.oligos
GQY1XT001.F003D146.sff GQY1XT001.oligos
GQY1XT001.F003D148.sff GQY1XT001.oligos
GQY1XT001.F003D150.sff GQY1XT001.oligos
…

Then run the following:

mothur > sff.multiple(file=sfffiles.txt, processors=2)

3

Many thanks, Sarah.

Three more questions about this.

  1. For one set of sequences, I sometimes have two barcodes per sample. Do I just give these different sample names in the oligos file and merge them after processing?
  2. For my second set of sequences, I have multiple samples per barcode. What to do there?
  3. I could still merge.sfffiles first for all the samples first and then run through the commands as in the Schloss_SOP–the oligos file would keep the samples separate, correct?

Thanks,
Beth

4

Three more questions about this.

  1. For one set of sequences, I sometimes have two barcodes per sample. Do I just give these different sample names in the oligos file and merge them after processing?

Mothur allows you to assign multiple barcodes to the same sample name.

Something like this:

forward CCGTCAATTCMTTTRAGT
barcode AATGGTAC F003D002
barcode AACCTGGC F003D002
barcode TTCGTGGC F003D004
…

  1. For my second set of sequences, I have multiple samples per barcode. What to do there?

Are the sequences in different files? Mothur allows you to assign a barcode to one sample. If you did something like:

forward CCGTCAATTCMTTTRAGT
barcode AATGGTAC F003D000
barcode AATGGTAC F003D002
barcode TTCGTGGC F003D004
…

Using a single oligos file, mothur would assign all sequences with the barcode AATGGTAC to F003D002. The only way around this is to have the various samples be in different files. For example is all the sequences from F003D000 are in one file and F003D002 are in another, then you can run the file for sample F003D000 with an oligos file like:

forward CCGTCAATTCMTTTRAGT
barcode AATGGTAC F003D000
barcode TTCGTGGC F003D004
…

And sample F003D002 with an oligos file like:

forward CCGTCAATTCMTTTRAGT
barcode AATGGTAC F003D002
barcode TTCGTGGC F003D004
…

Mothur would create a group file for each run that assigns the sequences to the separate samples even though they have the same barcode.

With sff.multiple you would just need multiple oligos files.

sfffile:

GQY1XT001.F003D000.sff GQY1XT001.1.oligos
GQY1XT001.F003D002.sff GQY1XT001.2.oligos
…