Hello,
I am currently trying to make.sra files from both Illumina MiSeq 16S data and from 454 ITS data (separately). A few questions have come up.
For the fastq files from the MiSeq 16S data:
I first tried to make a new fastq file from the contigs I’d made with make.contigs and my forward and reverse fastq files (with reverse complement oligos):
make.fastq(fasta=Reads_R1.trim.contigs.fasta, qfile=Reads_R1.trim.contigs.qual)
but this
make.sra(fastq=Reads_R1.trim_trial.contigs.fastq, oligos=oligos_16SMiSeqv3.oligos, project=test.project, mimark=MiMarksReads_R1contigs.txt, platform=ILLUMNA, instrument=Illumina_MiSeq, includescrap=FALSE)
put everything in scrap. I also tried different versions of the oligos (i.e. not reverse complement) and calling orientation=reverse, but this didn’t work either–everything still ended up in scrap.
Then I tried to use a file that specified the reverse and forward fastq with the same reverse complement oligos that I used in make.contigs:
make.sra(file=trialFandR.file, oligos=oligos_16SMiSeqv3.oligos, project=test.project, mimark=MiMarksReads_R1contigs.txt, platform=ILLUMINA, instrument=Illmina_MiSeq, includescrap=FALSE, pdiffs=2, bdiffs=1)
This seemed to work, slowly, but produced a new fastq file for forward and for reverse for every sample. It did not appear to scrap everything, but it did also did not process all the samples and specifically ended with supposed output files:
Output File Names:
trialFandR.Az_T165_526_AGGCTGATTTGC.forward.fastq
trialFandR.Az_T165_526_AGGCTGATTTGC.reverse.fastq
I weirdly cannot find these output files that make.sra has supposedly made.
and the command also ends with errors like:
[ERROR]: MIMarks file is missing group Az_T165_526_AGGCTGATTTGC, please correct.
My MiMarks file has group names (sample IDs) without the associated barcode obtained from get.mimarkspackage (e.g., Az_T165_526), is this not correct?
For the sff files from the 454 ITS data, I am not getting an errors but am having similar problems finding the output files, which I believe as a .fasta and .qual file for each sample were also already made when I originally ran sff.multiple to analyze the data? (What I am finding is the files of same name that I created in February 2015 when I began this analysis, and maybe I can just use those for the SRA data submission anyway.)
Sorry so much confusion, thanks for your help,
Beth