[rudi283]

Hi,
Have you tried to multiplex more than 4 samples?

[HGENETIC]

We have used the Nimblegen solution based capture method on a target region of ~1.5Mb. Our first expt screwed up fantastically well so we've had to repeat everything from scratch and are in the process of performing the QC on our results. We did the expt in 2 ways, we chose 8 individuals, sonicated and end repaaired etc, ran our precapture PCR to introduce barcoded primers then used qPCR to quantitate the samples and added eqimolar amounts of each sample to 1 hybridisation reaction. This was subsequently cleaned and prepared for sequencing as normal. The second method involved taking the samples through the whole protocol individually and only introducing the barcodes during the post hybridisation PCR, again the samples were quantitated using qPCR and then pooled together before sequencing. The thing about our protocol is that the majority of it is automated, all the cocktail addiditions and cleaning steps (using Ampure XP) are performed on a biomek fx and nx robot meaning in theory we can process 96 samples at a time. I'll have some results over the next week or two if anyone is interested?

[rudi283]

Have you tried an Array-Based Capture as well?

[HGENETIC]

No we've only used solution based capture

[rudi283]

right,
but if you could let us know anyway how did it work would be great
thanks