sample prep kit recommendation?

[jwa009]

Hi I am new to NGS. and I have read a lot from your posts. thanks first.

we plan to do NGS from dsDNA for target exome sequencing. the start sample could be 1ng to 1ug, in about 10-20ul.

I now need your advice: which sample prep kit I should pick as so many are out there.

we have Covaris for fragmentation. so mechanical way is not a problem. I do heard nextera can simplify workflow but there is concerns for bias. I personally think nextera is good for low input samples. so I will use standard lib prep workflow first.

I get lost which kit to use. TruSeq DNA Prep kit was said good, but we are hesitated because there are 3 purification steps before lib amplification. And NEB and Kapa all have 1 purification workflow before PCR.

However, what I will miss if i simply choose NEBNext Ultra kit or Kapa Hyper Plus? And is there other kit as better choices? Less cost and good quality are the two main concerns for us.

appreciate your response.

[nucacidhunter]

I think first one you should decide what region is appropriate for the study aims. There are various off the shelf choices for some species and custom ones also can be ordered. The content of target region among kits varies. Major hybrid capture kits includes those from Agilent, NimbleGen and Illumina with first two having number of options. Most kits are sold with library prep reagents. If available DNA amount is not sufficient for the chosen kit, there are options for preparing libraries with third party kits then using those as input for hybridisation. There are also PCR and restriction enzyme based kits. You can find papers comparing different exome capture platforms. Here is a paper evaluating few library prep method on capture. http://journals.plos.org/plosone/art...l.pone.0101154

[jwa009]

thank you so much.
I got your point that we should find right target pull down strategies before making decisions.
also appreciate your link for the paper. reading now.

[nucacidhunter]

If you could give more info on species and study aim I may be able to narrow down available kits. Note that some exome capture kits only provide baits for exome (the content can vary from vendor to vendor) and others for exome plus UTR, lncRNA or option of adding custom target region to pre-manufactured exome baits. Most of standard kits require input DNA in excess of 50 ng. For inputs less than 50 ng one has to use third party kits for pre-capture library prep.

[kushald]

Have you tried the Ion Ampliseq Exome on Proton using the Hi-Q chemistry. gDNA samples input from as low as 10ng and has higher on-target coverage compared to conventional kits. The new P-II chip on Proton can give as much as 24GB of data in a single run.

[kyrial]

We have had really good success with the Kapa Hyper library prep kit for low input (or standard input) samples, then taking those libraries into an Agilent XT capture. One trick we have found to help with this process is to increase the amount of XT adapter used in the library ligation step (we currently use a 1:100 insert to adapter ratio instead of the typical 1:10 ratio).