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Old 09-27-2016, 12:44 PM   #5
Yepler
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Location: Tucson, AZ, USA

Join Date: Oct 2010
Posts: 22
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Sorry it took me a few days to check back. I might have identified your problem, although it might also be colored by my own experiences.

I have quite small-insert libraries, and while they are homemade, they come out looking like standard Illumina dual-sided barcoded libraries. The only real difference is that the main read is sequenced with the Small RNA Sequencing Primer (from Illumina), which has a Tm of about 69 C, while the other Read1 primers in Illumina's mix have Tms around 74 C. When I tried sequencing my libraries on the NextSeq, what I saw was two lanes of decent sequence and two lanes of all Gs. If you know the NextSeq, you know that all Gs means (basically) that the cluster didn't sequence. But the cluster itself DID demultiplex, so the two index reads were successful.

I should point out here before someone asks - yes, I'd sequenced these libraries extensively on MiSeq and HiSeq prior to my NextSeq Adventures. I do a lot of sequencing! Anyhow, after a lot of back and forth with Illumina, we finally hit on the Tm of the Small RNA Sequencing Primer.

I couldn't make the primer longer, because there wasn't any extra room, so instead, I added LNA bases to bring the Tm up to ~74 C. I added the LNA primer to the Read1 custom position, and out came some gorgeous sequencing.

So...I quickly put your primer sequences through IDT's Tm calculator, and the Tms are pretty low. Can you increase them, either via length, or via LNAs, to nearer to 74?

I may be off the mark here, so feel free to send more details back. But after my headache, I'd look at Tm first.

Oh, and the other thing that might be helpful is to make sure that a "standard" library (you could use PhiX, in a pinch) DOES sequence well. I was lucky enough to have a very similar library that used the standard Read 1 primer, and that helped narrow down the issue.
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