Dr. Prepper

NOTE:  This is a repost of an entry that I wrote for the molecularecologist.com.

Library preparation for next-gen sequencing has become a fact of life in many labs working with model and non-model organisms. The problems with library preparation are that (1) library prep is slow and (2) library prep is expensive. Generally speaking, you can improve #1 OR you can improve #2, but you can't do both simultaneously.

Most of us can justify time in the lab as long as we can reduce per-sample costs, so I wanted to quickly present a few options for library preparation along with their per-sample (list price/units) cost. In the following, I'm going to focus on the exact "kits" that you would use to prepare DNA libraries for Illumina sequencing using the TruSeq (Tm) adapter/reagent system:

Illumina TruSeq DNA Sample Prep v2 (kit cost $2,600; 48 samples; $54.17/library - these are the standard Illumina library prep kits - meaning that these are the kits supported by Illumina for use on their machines. These kits also include adapters incorporating sequence tags (n = 24) to identify reads in multiplex. The other kits below do not - meaning that the cost of adapters is a cost you will need to cover. If proper customer support and guaranteed performance interest you, then this is your kit.

NEBNext DNA Sample Prep (kit cost $1,400; 50 samples; $28/library) - this is a handy master-mix kit that includes everything you get, above, except for the adapters incorporating sequence tags. Comes with a decent set of instructions and nicely-priced. All reagents included in kit are validated (in batch) by prepping an DNA library prior to shipping.

Kapa Biosystems Library Preparation (kit cost $1,120; 50 samples; $22.4/library) - a new and welcome entry to the library prep kit selection that is similar, in content, to the NEBNext kits. The Kapa kits uses Kapa's proprietary HiFi HotStart mix, which has been show to produce libraries with lower amplification bias**. Does not include adapters incorporating sequence tags.

If you're wondering about the cost of sequencing adapters including identifying tags, those are generally a relatively high fixed-cost. But, once you buy adapters, you tend to have sufficient amounts to prepare many, many libraries (i.e., many more than 48 or 50). There is additionally some debate, when you purchase adapters, about whether or not you should have them HPLC-purified [more info on what HPLC does] and/or whether or not the universal adapter and PCR-primers should include phosphorothioate bonds at their 3' termini [more info on what phosphorothioate bonds do]. I would suggest that you do both, particularly using HPLC in combination with certain types of sequence identifying tags (a post for a later date):

Cost of 25 (24-tagged; 1 universal) HPLC-purified, TruSeq-style adapters at 250 nmole: $2,875 ($115/adapter). Price estimates from idtdna.com.

Finally, if you are in a hurry, you want an easy and FAST way to prep your libraries, and/or you have limited amounts of template from which to build libraries (>= 50 ng), you should consider:

Illumina Nextera DNA Sample Preparation (kit cost: $2200; 24 samples; $91.67/sample) - these are the Cadillac of library prep and are worth their weight in gold if you are time or DNA limited. We've used these on all kinds of DNA, from fresh material to museum samples, and they've worked consistently and well. You can multiplex up to 384 samples using a dual indexing strategy that is provided with the new (Illumina, not Epicentre) kits.

In summary, if you want an all-in-one package that contains the reagents you need, promises full customer support, and you have relatively few libraries to prepare - you may want to go with the standard Illumina TruSeq library preparation kit. If you are comfortable with library preparation, you have lots of libraries to prepare, and you need to cut costs, look into the NEB or Kapa kits - both are very nice. Finally, if you are template-limited, in a hurry, or want to minimize processing steps, I'd suggest the Illumina Nextera kits.

**Kapa also offers an interesting all-in-one library prep kit that includes qPCR reagents to help you determine the optimal number of amplification cycles to use during library preparation.


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