![]() What I'm showing here is the deck layout that's required by the method. The protocol in question was automated on our Biomek 4000 liquid handler, which is the smallest of our Biomek platforms, largely by Sarah Simons, one of our very capable field application scientists. I am going to talk about some of the work that we'd performed here at Beckman Coulter to automate this protocol. I am going to, I think, pass control back to Zach. It's easy, and it produces very high quality Ion Torrent libraries, and so thank you. In our kit, we also have a protocol for read size selection for libraries, and this would give a much broader size distribution, and so we think we have a kit that has a streamlined workflow. It shows very high matching to the genome and good quality. This was a 200 base pair library that we size selected on E-Gel, so you can see that the read length distribution is quite tight and the data that was produced, it's very high quality. In addition, the libraries that are generated with the kit produce very high quality sequencing data, so I'm showing you a snapshot of one of our run reports for our library, one of our libraries. The gel was going for a little bit longer and then the 400 base pair library is eluted. The 200 base pair size library was eluted first. Then, it was run on an E-Gel to size select. I'm also showing you that the kit can be used to construct libraries of different sizes, so in this particular library construction, a single prep was made for each library. This kit is also compatible with a broad range of inputs, so I'm showing you libraries that were constructed from DNA isolated from some different organisms, and the GC content in these organisms range from 38% to 65%. The kit is compatible with a broad range of inputs from 10 nanograms to one microgram, and it's compatible with multiplexing, and as I said, PCR free library construction. It takes about two hours to make a library, and there's very little hands-on time involved. We have a simple protocol with a streamlined workflow. We fairly reached our goals with this kit. We've also combined the ligation as well as the nick translation step, and that allows for the construction of PCR through the libraries, and so after the ligation and nick translation step, the sample is cleaned up and size selected, and then the sample can either be sequenced directly or it can be enriched in PCR amplification. We've also had this particular cup is able to be inactivated with heat, and so that removes the need to clean up the sample after this step. This allows us to use, as input, intact DNA. The kit we developed is called the NEBNext Fast DNA fragmentation and library prep set, and what we've done with this kit is we've combined the fragmentation and end repair into one step. We also wanted to eliminate the need to fragment DNA before going into library prep, so we wanted to have, as our input, intact DNA, and of course, we wanted to create a low-cost solution for our customers. The goals we wanted to have were to develop a fast and simple protocol that would minimize hands-on time as well as the ability or the possibility of errors. When we were developing a kit at NEB, we had a number of goals that we set for our kit. In this system, the adaptors are not phosphorylated, and that aids in reducing the amount of adaptive diamond it's produced, but it causes an incomplete or results in an incomplete ligation, so there are nicks remaining in the DNA after the ligation that have to be repaired, and those are repaired by a nick translation reaction.įollowing the nick translation, the library is enriched by amplifying in a PCR reaction, and so there's a number of steps that occur during library construction, and after each one of these steps, there's typically a clean-up that occurs, and also after the ligation and nick repair, there's also a size selection and a repair and a clean-up. Those fragments are then end repaired to generate five prime phosphorylated blunt-ended molecules onto those molecules of ligated P1 and A adaptor. ![]() The first step involves fragmentation of the input DNA. I'm going to talk a little bit about one of the kits we developed at NEB for Ion Torrent library construction, but what I'd like to do is begin with just an overview of the workflow and describe the steps involved in generating a library for Ion Torrent sequencing. I'm having a little problem with moving the slides. I want to thank everyone for attending the seminar. With that, I would like to begin this talk by passing the presentation off to Dr. Lynne Apone from New England Biolabs, and we're very pleased to have them here. ![]() I'd like to welcome you to our online webinar about increasing productivity and reproducibility by automating DNA library prep for Ion Torrent sequencing. ![]()
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