Our lab has recently sequenced 16S using the MiSeq platform with primers that were originally created for GAIIx. The primers worked successfully for GAIIx but it appears that they may not have worked as well for MiSeq. The primers include the illumina sequencing primers instead of custom primers.
here is the GAIIx forward primer sequence order:
Illumina adapter - linker - barcode - Illumina Sequencing - 16S primer
CAAGCAGAAGACGGCATACGA - GAT - AAGGTGG - GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT - AGAGTTTGATCCTGGCTCAG
We sequenced the V1-V2 region. We only put barcodes with the forward primer.
The problem that we are seeing is after we join the two reads with make.contigs and then trim off the barcodes with trim.seqs. Only about 10% of our reads seem to have the barcodes (which seems very low to us, especially compared to our GAIIx results). Also, the reads appear to have different parts of the illumina sequencing primer still attached to the forward 16S primer.
Here are some sequence examples:
TGCTCTTCCGATCTAGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCCTAATACATGCAAGTCGAACGCCCC…
TTCCGATCTAGAGTTTGATCCTGGCTCAGAACGAACGTTGGCGGCACGCCTAACACATGCAAGTCGAACGAAGCAACTG…
CGTACTGGCAGACTTGCTCTTCCGATCTAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGC…
Has anyone else seen these issues when using the illumina sequencing primer with MiSeq? Is there a potential work around that I can use with mother to get rid of these illumina sequencing primer parts? (could these also hinder mother from being able to find barcodes on some sequences?)
Thank you very much!