Hello!
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
forward GTGTGYCAGCMGCCGCGGTAA
#reverse CCGGACTACHVGGGTWTCTAAT
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?
Thank you!