HaloPlex for library preparation

[ycding]

HI, I wonder whether anyone has used HaloPlex tech to prepare library for illumina highseq 2000 sequencer. does it work well for you?

many thanks, ding

[rainbowboron]

I would also be interested in hearing what others have to say as well! I just got a HaloPlex kit, ran through it once... and bam! Nothing.

[henry.wood]

ran through it once... and bam! Nothing

My bams had something, but not as much as they should. I asked a question about this a few weeks ago but got no response. We sequenced two lanes on a HiSeq and they were fine, but a third and fourth one had very low number of reads passing filter. At the moment our best guess is that the lanes were overloaded. We massively reduced the loading density for the failing samples and it seems to be working, although we are still testing things. Maybe there is something about haloplex libraries that makes them difficult to quantify accurately for the purposes of optimal loading. If anyone has an alternative theory I'd love to hear.

[barrmur]

Haloplex libraries

I have just completed a run of a FC using the Haloplex exome kit. While the protocol is nice and easy the cluster numbers are confusing me a bit. We quantified initially using the bioanalyzer and loaded our pools at the standard 8pm that gives us good cluster numbers on the hiseq (about 800K/mm2). These were down around the 300K mark.

The FC crashed so we ran again with a 12pm loading (I was slightly worried about overloading so aired on the side of caution) and we got around the 500K mark. I have since gone back to my pools and quantified by qPCR using the KAPA kit and the results were pretty similar to the BA. Not really sure what is going on.

We have also just run some sure select libraries using the BA to quant and loaded at 8pm resulting in good numbers. Halo libraries just dont seem to cluster as well as we had hoped. Running more soon so I will keep you updated.