help with make.contigs/illumina MiSeq data

Commands in mothur
1

Hello,
Having a novice problem. From my sequencing provider I was given three fastq files from our run: read1, read2, and index. I was also given a mapping file with the barcodes since my samples are only a subset of the run. At the mothur course and in practice until now I have only used data sets where there is a separate R1 and R2 fastq for each file. I am having trouble getting past the first steps. I have tried to run “make.contigs(ffastq=yourForwardReadFile, rfastq=yourReverseReadFile, oligos=yourOligosFile)” but everything ended up in the scrap file and the trim file was empty. I was wondering if I am setting up my oligos file wrong and I wasn’t sure which sequences I should be putting in the oligos files aside from the barcodes. I had tried with just the 515 forward primer and reverse primer but this failed. Do I need to do anything with the Illumina adapter? Here is what additional information I have:

5’ Illumina Adapter
AATGATACGGCGACCACCGAGATCTACAC
Forward Primer Pad TATGGTAATT
Forward Primer Linker GT
515 Forward Primer GTGCCAGCMGCCGCGGTAA
Primer For PCR AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA

Forward Primer Pad
TATGGTAATT
Forward Primer Linker
GT
515 Forward Primer
GTGCCAGCMGCCGCGGTAA
Read 1 Sequencing Primer
TATGGTAATTGTGTGCCAGCMGCCGCGGTAA

Reverse Primer Pad
AGTCAGTCAG
Reverse Primer Linker
CC
Reverse Primer
GGACTACHVGGGTWTCTAAT
Read 2 Sequencing Primer
AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT

RC of Reverse Primer
ATTAGAWACCCBDGTAGTCC
RC of Reverse Primer Linker
GG
RC of Reverse Primer Pad
CTGACTGACT
Index Sequence Primer
ATTAGAWACCCBDGTAGTCCGGCTGACTGACT

If I could get this to work I was hoping to then make.groups to then pull out just my samples. Is this the right approach?
Did I understand right that version 1.33 is going to allow us to pull out the fastq files for a subset of samples with fastq.info command? Will that be another way to get out of this jam?
Thank you so much for help with this question, sorry if it is really basic.

2

I am having the same issue! I signed on to the Mothur forum to post an inquiry about this but saw yours and decided to wait for someone to reply to yours. Looking forward to a reply with some guidance on how to resolve this issue,

3

Can you post a few forward reads and reverse reads as well as the oligos file you created so we can try to track down the issue?

4

Sorry for the delay, I was off the grid. I think the more recent thread “help with make.contigs using an index file” may fix my problem so I am going to try that first and then will get back if there are still problems.