make.contigs w/ oligos file

Commands in mothur
1

Hello,

The previous mothur version I was using did not have the oligos option, so I am hoping to get clarification on this. I have fastq files that have barcodes removed but presumably still have the 16S V4 primers attached. The program I use to demultiplex can trim primers but only based on primer length, so I opted out based on that seeming a little cruder than I’d like. I decided to take advantage of the oligos option in make.contigs but I’m confused on the right setup. First I made a .file with two columns:

C0_A_R1.fastq.gz C0_A_R2.fastq.gz
C0_A1_R1.fastq.gz C0_A1_R2.fastq.gz


Then my oligos file is formatted for pcr.seqs as such:

forward GTGYCAGCMGCCGCGGTAA
Reverse GGACTACNVGGGTWTCTAAT

When I ran

make.contigs(file=oil_sed.file, oligos=oil_sed.oligos, processors=24)

I got the following error:

make.contigs requires paired barcodes and primers. You can set one end to NONE if you are using an index file.
Am I required to supply barcodes (unnecessary since mine have been removed)? If I don’t need barcodes, I am thinking I need to format a specific oligos file for make.contigs which looks like this:

primer GTGYCAGCMGCCGCGGTAA GGACTACNVGGGTWTCTAAT

Thanks!

2

I went ahead and attempted

make.contigs

with the .file and latter .oligos format above. With the two column .file format, I didn’t get a .groups output. So, I ran

make.contigs

with the latter .oligos file and a 3 column .file as shown in the mothur wiki. This time I got a .groups file from my output, but I am still unclear on whether the .oligos file I provided removed my forward and revers 16S primers.

Thanks!

3

If you run “head” on the first few lines of the outputted fasta file, do you see the oligos? If they made it through the command then they should have been found and removed.

Pat

4

Hi Pat,

I am assuming that if they weren’t removed I should see them either at the beginning or end of the sequences in the contigs.fasta. I am not, so they must have been removed?

Thanks!

5

Correct - you might double check the reverse complement of the oligos too, but I think you’re good to go.
Pat