I have sequenced samples using Miseq and I want to analyse them using mothur. I read the Miseq Sop and also another topic in this forum (demultiplex dual index fastq files). As I can see, I need to demultiplex my fastq files and use them to make contigs using the make.contigs command, as described in the SOP.
I´m trying to use the fastq.info command to demultiplex my sequences, but all sequences are falling in the scrap files. In another topic I read that I need to use the command with only the forward or the reverse primer, in the respective fastq file containing all sequences. I´m not sure how the oligo file must be…I have used mothur to analyse 454 data before, and also Ion Torrent.
Below are the first lines in the oligo file. When I use the fastq command I remove the forward or the reverse primer sequence, as described in the other topic
barcode TCGGATCTGTGA 10C barcode GCACAAGGCAAG 29C barcode CTCGGATAGATC 42C barcode CGATATCAGTAG 13Me barcode CTTGCGGCAATC 21Me barcode CGTTCTGGTGGT 34Me barcode TGCGGTTGACTC 8MeC
The command I´m running is this: fastq.info(fastq=S0_L001_R1_001.fastq, oligos=oligo.oligos, pdiffs=2, bdiffs=1, fasta=t)
Could anyone help me with this? What am I doing wrong? Do I need to include also the linker sequence?