Ion Torrent targeted sequencing

[MonaE]

Hello Every one,

I am a new NGS user. I am starting to work on targeted sequencing using Ion Torrent technology.

My targeted gene is BRCA1 and I have about 50 samples. The gene is 112 amplicon. I would like to know how many samples per run I can pool?

Thank you for your help

[jimmyinfrance]

That depends a little on your experiment, ie how big your amplicons are, if its germline somatic mutationd and which chip you want to use ie 314/316/318.
FYI -- we have recently been testing Sabioscience (Qiagen) gene-read (multiplex PCR) for this type of targeted experiment, seems to work well. We estimate (guess-timate) that 48 samples should be reasonable on a 316 (200mbp) Ion run.

[MonaE]

Thank you for your replay.
I was just wondering how do you do these calculations?

I found out that if I used 318 I will get a coverage of 7,84 but should use tow chips as the chip takes 48 samples. on the other hand if I used 316 I will have a coverage of 1,57. My samples are DNA from blood, do I need still (big) coverage?

[jimmyinfrance]

Here's a back-of-the-envelope calculation (i.e. it's rough).

Assuming that average amplicon size = 200 bp
you have 112 amplicons,
you want 100X seq coverage

= 2,2 mbp/individual (ie 200bp X 112 amplicons X 100 fold).

2,2 X 48 = 107 mbp total. ABout capacity of a 316 mbp

Very rough though so maybe overestimate needed coverage in order to achieve the minimum you need (ie difference in amplicons, barcodes etc).

[MonaE]

Thank you