A collaborator recently sent 88 samples off for sequencing. What she got back were 4 fastq files - SAM1-44-r1.fastq, SAM1-44-r2.fastq, SAM45-88-r1.fastq, and SAM45-88-r2.fastq. For each of these 88 samples, we know which barcode was used (the same barcode was used for both forwards and reverse reads). Initially I thought that we would have a huge scripting job to parse the samples out into individual forward and reverse fastq files for the MiSEQ SOP in mothur, but after reading through the make.contigs description on the wiki it seems like I should be able to use the oligos option, sending the forward and reverse fastq and the oligos with the barcodes in it. Is that correct?
The next concern I have is that I am not sure how to make this all work with the reads split up (SAM1-44 and SAM 45-88). Do I need to concatenate the forward reads together, as well as the reverse reads, so that I can send make.contigs just the two fastq files?
Alternately - the service my collaborator did provide a fasta file containing the already-assembled contigs. I’m just not sure if I can insert this into the MiSEQ SOP, since I won’t be getting any groups information this way.
Help?
A collaborator recently sent 88 samples off for sequencing. What she got back were 4 fastq files - SAM1-44-r1.fastq, SAM1-44-r2.fastq, SAM45-88-r1.fastq, and SAM45-88-r2.fastq. For each of these 88 samples, we know which barcode was used (the same barcode was used for both forwards and reverse reads). Initially I thought that we would have a huge scripting job to parse the samples out into individual forward and reverse fastq files for the MiSEQ SOP in mothur, but after reading through the make.contigs description on the wiki it seems like I should be able to use the oligos option, sending the forward and reverse fastq and the oligos with the barcodes in it. Is that correct?
The oligos option should work for you.
The next concern I have is that I am not sure how to make this all work with the reads split up (SAM1-44 and SAM 45-88). Do I need to concatenate the forward reads together, as well as the reverse reads, so that I can send make.contigs just the two fastq files?
I would concatenate the fasta and group file after you’re done with the preliminary processing (i.e. before running unique.seqs)
Also, the contigs we’ve seen generated by other methods are generally not as good (i.e. higher error rates) than those generated by make.contigs.
Pat
Thanks, Pat - this does seem like the easiest/best approach.
Quick question re the OLIGOS file format. I have the following sample information for each sample:
#SampleID BarcodeSequence LinkerPrimerSequence
Sample 1 CTTGCTTC AGRGTTTGATCMTGGCTCAG
Sample 2 CTTGGGCG AGRGTTTGATCMTGGCTCAG
Sample 3 CTTGATGT AGRGTTTGATCMTGGCTCAG
The same barcode is used for both forward and reverse reactions, so can I just create the following for Oligos, using the reverse complement of the barcode for the second barcode? Or do I not need to do the reverse complement?
#My Oligos File
BARCODE CTTGCTTC GAAGCAAG Sample1
BARCODE CTTGGGCG CGCCCAAG Sample2
BARCODE CTTGATGT ACATCAAG Sample3
so can I just create the following for Oligos, using the reverse complement of the barcode for the second barcode? Or do I not need to do the reverse complement?
You’ll definitely need the second barcode, but I think you’ll want the normal primer sequence since mothur reads the sequence in the direction it was created. If this doesn’t work, try the reverse complement and let us know on this thread.
Pat