I don’t understand how I can prepare data for submission to the SRA public repository. The problem is that the MiSeq run included both 16S and ITS sequences, and I only want to publish the 16S data for now (hopefully, the ITS will come later on).
The original data are in a collection of 2*48 fastq files (1 file for forward, 1 file fo reverse, 48 samples), each fastq file comprising both 16S and ITS sequences. To do my analyses, I separated the 16S and ITS sequences with pcr.seqs (and an oligos file) right after making the contigs.
Can I just run make.sra with both file and oligos options? Will mothur understand that the reads have to be assembled into contigs?
Thank you for your help,
I understood that I can’t have both the 3-column file and the oligos options. But then, how can I select only the 16S sequences and exclude the ITS? That’s what I tried the oligos file option for (with PCR primers, not sample barcodes, because the samples are in separate fastq files).
Can you try going back to the original fastq files and using remove.seqs to remove the ITS reads before running the make.sra command?
mothur > remove.seqs(fastq=file1.forward.fastq, accnos=.bad.accnos) - the bad.accnos file should contain the list of sequences removed by the pcr.seqs command.
mothur > remove.seqs(fastq=file1.reverse.fastq, accnos=.bad.accnos)
mothur > remove.seqs(fastq=file2.reverse.fastq, accnos=.bad.accnos)
Then you can run the make.sra with a 3 column file:
sample1 file1.forward.pick.fastq file1.reverse.pick.fastq
sample2 file2.forward.pick.fastq file2.reverse.pick.fastq
sample3 file3.forward.pick.fastq file3.reverse.pick.fastq
Thank you Sarah!