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TruSeq RNA Sample Prep Kit v2 – First Impressions/Initial Review

January 1, 2012

I decided to get the TruSeq RNA Sample Prep Kit v2 to construct a bunch of RNA-seq libraries. I decided to go with it because it is well priced and has gotten some good reviews. It also relives the logistics of maintaining all the reagents for a ‘homebrew’ kit. Unfortunately, it was backordered and I was initially told it would take two weeks to arrive, which turned into two months. Usually, I associate these types of delays with doing science in Greece but this time I am pretty sure it was Illumina to blame. Anyway, the kit finally arrived and my first thought is that the people that wrote the manual need a serious lesson in scientific writing. With all the acronyms used throughout the manual it is nearly impossible to read. This is lesson one in scientific writing, avoid acronyms and jargon whenever possible. There is also a typo in it, 79.6 μl is written in a few places where it should be 9.6 μl.  I’m sure there are some people out there looking for a 4 ml tube of First Strand Synthesis Buffer when only 460 μl is provided. However, since I am the undisputed king of the dyslexic typo, I’ll let their writers slide on that one. Even the ‘Experienced User Protocol’ was too wordy and cryptic to follow, so I translated the protocol into plain English, which can be found at: TruSeq RNA Sample Prep Kit v2 protocol translated into plain English – caution it may contain typos but probably less than Illumina’s version.

I processed two samples to get a feel for the kit, as I didn’t want to blow the whole thing at once and not have it work. It appears (the samples still need to be sequenced) to work really nicely except for their recommendation of 15 cycles of PCR. It seems plain stupid to recommend a specific number of PCR cycles. The optimum number of cycles is clearly dependent on quantity, quality and composition of the starting material. Against my better judgment, I followed their protocol to the letter and my library was over-amplified. I got the craziest bioanalyzer image I’ve ever seen!!!! I really have no way to explain it. Anyway, I took 1 μl of my library and subjected it to 5 cycles of PCR with the Kapa SYBER Fast polymerase, purified it with the Qiaquick PCR purification columns and loaded that on a bioanalyzer DNA 100 chip and the size distribution looked much better.  There is still some larger material, which I’m not sure what it is. I don’t think it is Ampure XP bead carryover, so perhaps it is DNA molecules annealed at their adapters. The Bioanalyzer DNA1000 images are below.

What I would like to see in the kit that it does not have:

1. dUTP/UDG strand specificity.

2. A better PCR polymerase, i.e. the Kapa polymerase.

3. Better information on how to determine the correct number of PCR cycles like I include in my ChIP-seq library construction protocol.

It seems entirely reasonable to halve the starting material and reaction volumes to get twice as many libraries out of the kit. When we are multiplexing 10+ samples in a lane, there is no need to generate so much library.

All in all I think you can put together a better kit with strand specificity for probably a little less money, but if this is your first foray into Next Generation Sequencing this kit is very easy and at this point appears to be a good option.

Update:  I have made about 30 libraries with the kit.  All of them went well, i.e. they looked nice on the bioanalyzer.  The 15 PCR cycles recommended by Illumina is clearly too many. I have been doing 10 which has been producing libraries with a concentration of about 150 nM, which is at the upper limit for the PCR amplification. Also, I have been using one-third the reagents of what is written in the Illumina protocol so that I can get 150 libraries out of the kit. The Illumina protocol produces about 30 μl of library at a concentration in excess of 100 nM, which is probably two orders of magnitude more than what I need.  I wrote up the protocol in a nice and easy format to follow and it is here:

TruSeq RNA Library Preparation Kit V2 using one-third the reagents


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  1. Are you going to do ScriptSeq and Ovation version 2 kits as well ? It would be neat if someone did replicates using different kits and published a platform paper. I know there was an article comparing some vendor’s version 1 kits published last year. I wonder how much better the improvements are.

  2. ethanomics permalink

    I’m really only into methodology in that it gets me to where I want to go, i.e. answer biological questions. Comparing methodologies for the sake of comparing methodologies is too boring for me. Anyway, the TruSeq kit has enough reagent for 50 samples and I see no reason why I can’t use 1/3 the amount of each reagent, so I can make 150 libraries with the kit. That should keep me busy for a while. I think after I am done with the TruSeq kit, I would like to use a dUTP/UDG strand-specific protocol.

    The ScriptSeq kit, I haven’t really heard anything positive about, so I wouldn’t spend my time or the labs money on it at this point. The Ovation kit is really expensive but you can use it for very small quantities of starting material. So I think in that case, if you can only get a nanogram of total RNA you go with the Ovation. If you can get a microgram you save the money and go with the TruSeq kit.

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