I’m a beginner with the analysis of Illumina data and I’m stuck at the very first step. It’s probably something easy to fix but I don’t know where to look at.And as I’m leaving the lab in less than a month, I’m a little bit in a hurry. I checked on the forum but I can’t find a solution …but all my apologies if I missed it !
My supervisor wanted an analysis with only single end data. I tried what was adviced on the Mothur forum (cfr. Illumina single-read with index in 2nd sequencing run) but unfortunately, I couldn’t manage to do the make.contigs. It never finds the paired read, even if the name are the same in the R1 file and in the rc-R1 file. I tried different parameters (reverse complement the barcode, changing the mapping file, using rindex, findex,…) but the problem is always the same.
This is an example of the cmd I used:
make.contigs(ffastq=sequences.fastq, rfastq=sequences.rc.fastq, oligos=oligos.txt, rindex=index.fastq, processors=8)
So I then tried to use the R1 and R2 files instead of the R1 and a rc file that I created myself. I thought it would be easier…but not really…There is an error message at the end is : Error reading quality file, name blank at position, -1 . The few sequences in the scrap files indicate a problem with the barcode.
This is an example of the cmd I used :
make.contigs(ffastq=sequences-R1.fastq, rfastq=sequences-R2.fastq, oligos=oligos.txt, rindex=index.fastq, processors=8)
And this is an example of the oligos file that I used…(one of the numerous oligos file)
BARCODE NONE ACTAGGATCAGT TM1.0
BARCODE NONE GCTCCTTAGAAG TM2.0
BARCODE NONE TCCCATTCCCAT TM3.0
BARCODE NONE TGGCGTCATTCG TM4.0
BARCODE NONE AATCCTCGGAGT MW1.0
BARCODE NONE CTGGACGCATTA MW2.0
BARCODE NONE ACCGATTAGGTA MW3.0
BARCODE NONE ATGTGCTGCTCG MW4.0
Pat Schloss advised me to use trim.seqs for the single-end analysis but I don't know how to insert my index file...(again, maybe something really stupid, so I apologize in advance if it is the case). So, that's where I'm for the moment ...You'll probably need to se one or another file, so just ask :)
Thanks so much for your help!