trim.seqs...

Theory behind mothur
1

I tried the command below to process the samples and mothur’s response to it was below as well. What went wrong?

mothur > trim.seqs(fasta=cecum_mucus_Joy.fna, oligos=cecum_mucus_Joy.oligos, qfile=cecum_mucus_Joy.qual, maxambig=0, maxhomop=8, flip=T, bdiffs=1, pdiffs=2, qwindowaverage=35, qwindowsize=50)

bdiffs is not a valid parameter.

The valid parameters are: fasta, flip, oligos, maxambig, maxhomop, minlength, maxlength, qfile, qthreshold, qaverage, allfiles, qtrim, outputdir, and inputdir.
pdiffs is not a valid parameter.

2

what is the version number at the top of the screen when you fire up mothur?

3

its mothur v.1.8
Last updated: 2/02/2010

4

To run trim.seqs with bdiffs and pdiffs you need a more recent version of mothur. We’re up to 1.12, which was released a couple of weeks ago. Let me know if you still have problems.

5

Thanks a lot. The command works now. Now the *.groups file is empty. Here is the input oligos file:

forward AGAGTTTGATCMTGGCTCAG
reverse GCTGCCTCCCGTAGGAGT
barcode ACGAGTGCGT E1
barcode ACGCTCGACA E2
barcode AGACGCACTC E3
barcode AGCACTGTAG E4
barcode ATCAGACACG E5
barcode ATATCGCGAG E6
barcode CGTGTCTCTA 1B
barcode CTCGCGTGTC 2B
barcode TCTCTATGCG 3B
barcode TGATACGTCT 4B
barcode CATAGTAGTG 5B
barcode CGAGAGATAC 6B

Does it look okay?

or is there something else wrong.

Here is the command:

trim.seqs(fasta=pyro_mic.fna, oligos=oligos.fna, qfile=3.TCA.454Reads.qual, maxambig=0, maxhomop=8, flip=T, bdiffs=1, pdiffs=2, qwindowaverage=35, qwindowsize=50)


Thanks a lot. Take care.
6

Progress! You probably want to put a # before reverse. The problem is likely that trim.seqs cannot find the reverse primer in your sequences. If the reverse can’t be found then it puts the sequence into the scrap file. I bet that if you look at the sequence names in your *.scrap.fasta file you’ll see a bunch of sequences that have something like >SequenceABCD|r.

The “r” means that it couldn’t find the reverse primer.

Hope this helps,
Pat

7

This helped a lot. Thank you for you time. Now the last problem (hopefully the last) is the *.groups file. It only found one of the barcodes. Is this a quality issue in the sequneces?

8

No problem - you might go back to the *.scrap.fasta file and look at the sequences where the name ends in “|b”. The “b” indicates that it couldn’t find the barcode. You might see how many of the sequences got rejected because they didn’t have the barcode and then look to see if you can eyeball the barcode yourself. It’s possible that you hit a jackpot with the one barcode or perhaps you have typos with the oligos file.

9

Thanks. I see lots of rejections. What is the meaning of “|n” and “|h” as lots of the headers contain that.

10

http://www.mothur.org/wiki/Trim.seqs

n - sequence rejected because it had an ambiguous base
h - sequence rejected because it had too long of a homopolymer region

11

Thanks for the help.