make.contigs

Commands in mothur
1

Hello,
I am trying to assemble my reads and also remove primers/barcodes etc. I used primers 515f (GTGCCAGCMGCCGCGGTAA) and 806r (GGACTACHVGGGTWTCTAAT) and index primers. How should I set up the oligos file such that the primers and barcodes are removed? Nothing I do seems to be working.

Thanks!!

2

Also want to mention that these reads are already demultiplexed!

3

Here’s a link to the oligos file page, http://www.mothur.org/wiki/Oligos_File. Since the barcodes are in the index file and the primers need to be removed here is the setup you are looking for.

Oligos file:

primer GTGCCAGCMGCCGCGGTAA GGACTACHVGGGTWTCTAAT
barcode forwardIndexBarcode reverseIndexBarcode barcodeName
morebarcode…
…

File File:
forwardFastqFile reverseFastqFile forwardIndexFile reverseIndexFile

mothur > make.contigs(file=file.txt, pdiffs=2, bdiffs=1)

NOTE: If you are missing an indexed barcode you can indicate it by using the keyword NONE.
ie. barcode forwardIndexBarcode NONE barcodeName