Nextera and multiplexing >12 samples?

[bcanton]

Hello SEQanswers,

I'm considering a MiSeq for verification of newly assembled plasmids (<10kb per plasmid). Obviously, I'd like to be able to multiplex many plasmids per run.

I know they sell a Nextera kit with 12 barcodes. Has anyone multiplexed ~96 samples together during Nextera sample prep? If so, how much of a pain was it?

Thanks!

Barry

[krobison]

I can't answer your question, but at $100/reaction, isn't this a pricey way to verify plasmids?

[bcanton]

Maybe I'm missing something but here was my thinking -

$1000/run ($750 for MiSeq run, a generous $250 for Nextera sample prep), ~100 plasmids multiplexed per run, so $10/plasmid with very deep coverage. For full plasmid sequence this compares pretty well with sanger at $3-4/read.

[epibio]

Has anyone multiplexed ~96 samples together during Nextera sample prep? If so, how much of a pain was it?

We've heard from several customers who are multiplexing 96+ samples using Nextera library prep without any problems. For an example using 96 barcodes, see Adey et al. Genome Biology 2010, 11:R119. The methods section of the paper discusses the design of the barcodes and the sequences are provided in the supplementary tables.

[krobison]

Maybe I'm missing something but here was my thinking -

$1000/run ($750 for MiSeq run, a generous $250 for Nextera sample prep), ~100 plasmids multiplexed per run, so $10/plasmid with very deep coverage. For full plasmid sequence this compares pretty well with sanger at $3-4/read.

But isn't Nextera $100 per library reaction? Or do you think you use much smaller amounts of the Nextera reagents than their manual specifies and thereby split it across the multiple plasmids?

Would certainly be cool if it works.

[ScottC]

This could also be of interest, although I'm not sure of pricing...

http://www.nugeninc.com/nugen/index.cfm/products/next-gen-sequencing/encore-384-multiplex-system/

[DZhang]

Image you have 100 plasmids in a 10x10 grid, you pool them in two dimensions so you have 20 pools with 10 plasmids each. So you only need 20 libraries. The tricky part is downstream informatics.

[larissa]

Image you have 100 plasmids in a 10x10 grid, you pool them in two dimensions so you have 20 pools with 10 plasmids each. So you only need 20 libraries. The tricky part is downstream informatics.

Something like this?
Citation: Neiman M, Lundin S, Savolainen P, Ahmadian A (2011) Decoding a Substantial Set of Samples in Parallel by Massive Sequencing. PLoS ONE 6(3): e17785. doi:10.1371/journal.pone.0017785