Issues with Fluidigm Access Array: Sequencing Multiplexed Amplicon Pools on MiSeq.

[_annaphase]

We are currently using Fluidigm Access Array to multiplex PCR for screening for mutations via MiSeq. We’re interested in talking to other groups that are using the Fluidigm Access Arrays to compare what they are seeing with amplicon dropout rates, designing amplicons for high and low GC targets, and regarding MiSeq performance.

We’re currently working with both Fluidigm and Illumina to try to resolve multiple issues. We’d love to hear from others how the Access Array is working for them for amplicon sequencing on MiSeq.

[Vinz]

We use Fluidigm extensively for amplicon sequencing on the MiSeq. And I would be happy to share experiences regarding quality if you have more specific questions.
Key to success is to keep primerdimer-rate low. We routinely get >80% Q30 if everything works well (600 to 800 K/mm2 for V2 chemistry).

[_annaphase]

Thanks so much for your reply, Vinz. Where do you guys get your FL1 and FL2 sequencing primers?

We are currently multiplexing ~480 amplicons per chip. Are you multiplexing at similar levels? About how many samples do you put per MiSeq run? We're happy to hear you guys are doing similar experiments.

[Vinz]

We use not exactly the outer primers that Fluidigm recommends. We changed it to work with the generic MiSeq sequencing primers. Full description is published here.
Multiplexing: 480 samples, 12 amplicons each for V2
768 samples, 12 amplicons each for V3

[luc]

Hi Vinz,

we are interested to use your protocol for other amplicons. For which melting temperature (IDT calculator) have you designed the sequence-specific part of your oligos?

Thanks in advance!

[Vinz]

We target an annealing temperature of 60°C.

[luc]

Thanks Vinz!