ultra-high multiplex pcr -custom method?

[oliver_ngs]

Hi,

I would like to use an ultra-high multiplex PCR method to create libraries for the Illumina Miseq.

My aim is to create a panel of 500 amplicons, that can be amplified in a single reaction, but I would like to avoid using a proprietory kit such as the IonAmpliSeq.

Does anyone have a custom ultra-high multiplex PCR protocol that they could share with me? The panel will be for routine work so I want to avoid using sequence capture methods.

Kind regards,
Oliver

[kwaraska]

Try this paper

Genome Res. 2012 May;22(5):939-46. doi: 10.1101/gr.128124.111. Epub 2012 Jan 20.

[timforshew]

Hi Oliver,

I have been using the Fluidigm system for this and it works well (I realise this isnt exactly what you are looking for but thought I would suggest it). I multiplex up to 500 amplicons for targeted sequencing of select genes. I perform the PCR using the Fluidigm Access Array. I then barcoded each of the patient samples individually. It costs about £4 per sample for the Access Array chip so not too expensive compared to other methods. I then run up to 384 patient libraries on a MiSeq lane (or HiSeq). Collegues have gone up to 1500 patients per lane so again this makes the sequencing pretty cheap. Just a thought.

Good luck and best wishes

Tim

[oliver_ngs]

Genome Res. 2012 May;22(5):939-46. doi: 10.1101/gr.128124.111. Epub 2012 Jan 20.

Thanks, interesting library multiplexing strategy.