I ordered 16S primers according to the earth microbiome website, however, the researchers added GCT to the end of the Forward primer Illumina 5' Adapter to affect the melting temperature of the primer.
My laboratory decided to use these primers with barcoded Reverse primers to reduce overall costs by using dual index instead of single index seq. A sequencing company has informed me that this "GCT" extension can cause issues as seen below:
"Reason being is that your P5 adapter flowcell binding site is 3bp longer than the standard Illumina adapter. As a result, all of the Index 2 reads will start with “GCT”. This low diversity region may end up with quality drop or even run stop"
Has anyone tried this particular this extension to the Illumina adaptor with dual indices, and not just single. The problem has not been flagged when the primers with the extension were used as single index barcodes, or by a different sequencing company so I do not know how serious this issue could be.
Thanks for your help
Kelly
Dear Kelly,
Yes, it could be troublesome, because the first three bases of i5 would be identical in each cluster, as your seq provider told you. High percentage of other library (with unrelated i5 sequences) could cure the problem though. I'd aim at no less than 20-25% addition of different type libs. Plus, these extra bases need to be added to i5 sequences in samplesheet, otherwise you will end up with all reads in the 'undetermined' file. You will also need to set the i5 length to 13 cycles to read entire barcodes (assuming that you are going to use the 10 bp Golay barcodes from EMP). Remember that MiSeq by default doesn't produce index reads fastq files, so no afterwards parsing is possible.
Best,
Marcin Golebiewski
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