Weird 16S custom primer issue on Aviti runs

[pmiguel]

Commonly, I mix pools from various projects on a typical 500 cycle MiSeq run. Specifically, I have groups submitting 16S amplicon libraries constructed using either the Pat Schloss laboratory protocol or a TruSeq adapter 16S protocol I helped design. The former requires custom sequencing primers, while the latter does not. However, if I add 3.4 µL of a 100 µM stock of the appropriate custom primer to the MiSeq slots containing the standard Illumina primers, everything works fine for both types of libraries.

When I attempt the equivalent process on the Aviti, it fails in a puzzling way: the Schloss-type libraries work fine, but the other 16S amplicon libraries are significantly suppressed (30-50 times lower percentage yield than on the MiSeq with the same superpool).

It took me a number of runs to pin this down, but having done so I can see why those 16S custom primers might interfere with the sequencing of the TruSeq 16S amplicons. Both the TruSeq and the 16S custom primers can anneal to the TruSeq 16S amplicons. My question is why doesn't this cause a problem on the MiSeq?

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Phillip

[IDDan]

Is it possible that the Aviti's annealing temperature is different than the MiSeq's (65C?)?

Is it possible that whereas the MiSeq's primer-wells allow segregation (or a 3primer--->4read run), the Aiviti doesn't (?), and as a result you have a new primer-primer interaction that you haven't had in the past?

Also, we've found that failure to do a buffer-swap after the odd gel-cut results in (Illumina/MiSeq) libraries not being melted by NaOH (buffered I presume), and on mixed runs the gel-cut variety simply drops out.

[pmiguel]

Is it possible that the Aviti's annealing temperature is different than the MiSeq's (65C?)?

Is it possible that whereas the MiSeq's primer-wells allow segregation (or a 3primer--->4read run), the Aiviti doesn't (?), and as a result you have a new primer-primer interaction that you haven't had in the past?

Also, we've found that failure to do a buffer-swap after the odd gel-cut results in (Illumina/MiSeq) libraries not being melted by NaOH (buffered I presume), and on mixed runs the gel-cut variety simply drops out.

Yes, that is likely that the Aviti's annealing temp is lower than the MiSeq's. But the Aviti has 4 primer wells and does 4 read runs.

About the gel-cut samples: did you denature your gel-cut libraries separately from your non-gel-cut libraries? I tend to make a giant pool of all the libraries and do a denaturation on that.

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Phillip

[IDDan]

Ah, four primer-wells on the Aviti. That's right. Thanks. Maybe if we get funds I'll get hands-on that platform.

IF it's a Tm issue, and NOT a primer-primer issue, then you might just LNA a few bases on your sequencing primers to increase TM without having to increase their lengths, IF you cannot increase length that is. That said, IF Element is copying Illumina, then their Tm is likely lower than MiSeq (60C; NovaSeq/HiSeq/NextSeq), so I would not expect it's a Tm issue, unless of course it allows a primer-primer interaction at say 60C that's otherwise prevented occurring at 65C, (or a primer-primer issue caused by mixing primers that were never together in an Illumina cartridge)?


RE the gel-cut samples, "usually" melted (NaOH denature) in parallel, not as a pool:
Not a hard and fast rule as to how we process gel-cuts vs. non gel-cut samples. Situation/concentration dependent, but, usually the different library-types (gel-cut (TAE IIRC) vs. bead-cleaned) were melted to 50pM separately, diluted to the loading concentration (9-18pM) in parallel, and then finally pooled at the "get to 600uL" stage just prior to loading on the instrument. We went through the generic "is my NaOH pH correct," step of troubleshooting the cluster-density issue. It was correct. We had initially processed pools in parallel with the only significant variable being the sample's buffer (and the gel-cut libs generate no clusters). We then reproduced that entire chain of events (including no gel-cut clusters), then buffer-swapped the gel-cut sample/pool (~1x beads ---> elute in EB/EBT or water), re-started at melt, and it magically worked (got gel-cut clusters). We stopped chasing that one (assumed the gel-buffer was buffering the NaOH) and implemented buffer-swaps after gel-cuts for Illumina. That issue never came back, not yet at least. note: Buffer-swap is also SOP after BluePippin for PacBio libraries, IF you gel-cut before library-prep that is, and I think they want you to buffer-swap if you Pippin after library prep (before ABC) also.

On MiSeq we never used the custom primer wells, instead adding our custom sequencing primers (CSP) directly to the stock Illumina sequencing primer wells. Did have to LNA our CSPs at one point, but that's another story involving a smart FSE/A, Illumina when it was young and hungry (flexible/reasonable), a de-tuned peltier, an eventually failed peltier, and my failing memory.