Your guide to navigating the challenges of library preparation for NGS; get through the process faster, more efficiently, and with fewer steps
Library preparation, being the first step in NGS workflow, is critical to the entire sequencing process and can significantly influence the quality and output of sequencing data; a low quality library often results in very poor data. Here we present some of the more common challenges you may experience during the library preparation phase of the workflow and potential solutions to avoid them.
Time is everything in the lab; the faster the protocol, the less likely you have to work late to finish the job and the more you can accomplish with your day. The typical library preparation protocol time can be upwards of 120 minutes just for the reaction (fragmentation, end repair & adapter ligation) to take place and can contain a number of hands-on pipetting and clean-up steps throughout the protocol. Not only does each of those steps potentially introduce bias, but really, who has the time? The Thermo Scientific MuSeek kits can shorten the time for library preparation to as little as 80 minutes, freeing up almost an hour for doing other things in the lab versus other kits. The Thermo Scientific ClaSeek kits can run in as few as 60 minutes in a PCR-free format!
It’s not just important to athletes and Gumby. Many kits are limited by the ability to handle only a single sample input amount (often 1 µg) and require processing it in a single manner; that’s just fine if you plan to do the same thing over and over again forever, but some days you need to try something a little different. That means you have to go buy and vet a brand new kit… or at least it used to. The ClaSeek kits are compatible with a number of different platforms and are incredibly agile; be it handling a range of sample inputs (from 5 ng – 1 µg) to handling both PCR and PCR-free protocols, this kit can do it all.
There is a unique frustration that results from performing an experiment and finding out only at the end that due to library preparation issues, the sample does not provide enough or accurate coverage in GC rich regions. By having the optional PCR step and allowing for variable sample input, the ClaSeek kits can help avoid this troubling issue by increasing coverage and thus ensure that the data accurately and reliably represents the biological sample.
Everyone has dealt with them at some point in their research career, and most people tend to find them more frustrating than enjoyable. Library preparation is often plagued by a significant number of hands-on steps, ranging from the physical fragmentation of the DNA sample to the many pipetting steps during end modification and adapter ligation. The MuSeek kit reduces hands on time and requires less pipetting through elimination of physical fragmentation, end conversion and ligation steps by using a transposon based system. The end result is a significantly simplified workflow that allows fewer opportunities for mistakes and provides a consistent high-quality library.