NEB TV, what's trending in science.
Welcome to NEB TV. Today, I'm joined by Andrew Barry, one of our product marketing managers. Hey Andrew.
Hey, Deana, how are you doing?
Good, thanks. And we're talking about target enrichment for next generation sequencing. In our Science in 60, Andrew will tell us a little bit about a new product from NEB, the NEB Next Direct Cancer Hotspot Panel, which utilizes a unique method for target enrichment.
Next, we'll interview Niall Lennon from The Broad Institute, and he will talk about some clinical applications for target enrichment related to oncology.
And lastly, we will speak with Isabel Gautreau, she's one of our scientists, and we'll talk about some tech support questions that she received on the subject.
Target enrichment describes a variety of approaches that have been developed to selectively isolate genomic regions of interests prior to next generation sequencing. While most hybridization based approaches first create a library out of the starting material, and then capture the material after it has been PCR amplified, NEB Next Direct first captures the material, then converts the captured material into a sequencer-ready library.
The technology works through hybridization of short biotinylated beads to each strand of genomic DNA that has been fragmented to a size of about 200 base pairs. The hybridized fragments are then captured using Streptavidin beads, and then converted to a sequencer-ready library through a series of enzymatic steps that remove off-target sequence and ligate universal adapters.
The final library contains two indexes: a sample index that allows samples to be pulled prior to sequencing, and a unique molecule index, or UMI.
The result is a one day protocol that enables the preparation of sequencer ready libraries with high specificity, uniformity, and sensitivity for the discovery and identification of nucleic acid variants.
The NEB Next Direct Cancer Hotspot Panel enriches 190 exons across 50 genes known to have implications across a range of cancer types.
Niall Lennon, PhD:
My name is Niall Lennon, I am the senior director of translational genomics. I'm product development at The Broad Institute of MIT and Harvard.
We're very fortunate at The Broad that our user community, our collaborators are based in our local hospital environment. And we are constantly striving to understand the clinical research that they're doing, and build and apply technologies to meet their needs as they hope to push forward their understanding of the molecular basis of disease, cancer specifically.
When it comes to next generation sequencing, you can decide to do a variety of different types of sequencing libraries. For cost reasons, you may decide that you actually don't need to sequence the entire genome, but really you want to only look at the expressed portion of that genome, that is the exome. There may be cases where even the whole exome is too much sequencing. For instance, in cancer, you actually may be more interested in having a high sensitivity to detect variants in a low allele fraction of tumor cells within a biopsy tissue.
So if you were doing, for instance, clinical diagnostics, you largely know the genes that you want to test, and you largely know the variants you're looking for, if you're looking to match a patient to a specific therapy, for instance, or enroll them on a specific trial. And therefore you may want to go with a targeted panel, and sequence that panel more deeply to have the optimal balance between cost and sensitivity. We've had several examples in our lab of individuals where tumor sequencing has predicted the type of therapy they should go on, or a clinical trial they should enroll in.
And for those individuals, that single sequencing, that single sample sequencing has been transformative in their lives.
One example of a study that I think is indicative of the way that cancer research is going is the metastatic breast cancer project, a project driven by Nick Wagley, a local oncologist at Dana Farber and The Broad. That project uses social media and patient outreach to enroll metastatic breast cancer patients, male and female, across the country who may not live close to an academic medical center, and who may otherwise have never had their own tumors sequenced. This is a very empowering act for many of them, and is a very powerful study design for the cancer community at large.
Other exciting areas in the future that we see are the application of liquid biopsies. That is the ability to detect tumor sample from a blood draw, as opposed to a traditional biopsy, tissue biopsy. This technique will allow us to track the evolution of tumors over time, to track response to drugs. We're very excited to start to work on blood biopsies at the institute. Something that's new for us is that we actually now, in the last couple of years, have started to offer many of our sequencing services, externally outside just The Broad community. All of our exome, genome, and panel work is now available as a service, as well as a collaboration.
Anyone who wants more information on that could go to genomics.broadinstutite.org, and there is a list of all of the available services there.
My name is Isabel Gautreau, and I'm a technical support scientist at NEB. I support the NEB Next Direct product line.
One of the questions I get from customers is, “What sequencing read length is the NEB Next Direct Cancer Hotspot Panel optimized for, and why is this important?”
The NEB Next Direct technology can be optimized for any sequencing parameters, but this particular panel was optimized for 2 x 75 base pair reads. This is because we use both strands, and the exons are 125 to 130 bases long. By sequencing 2 x 75 base pair reads, you'll have a little bit of overlap in the middle, and you don't lose any information due to having gaps.
Another question that we get from our customers is how important it is to avoid bead loss during the protocol, and what is the impact of having bead loss. It is very important to avoid bead loss because the beads are streptavidin modified, and the baits are biotinylated. This bond exists throughout the entire protocol. Because of this, we don't have to do any cleanup steps, and we don't have to do any transfers. We just do simple buffer exchange washes.
There are six exchange washes in the protocol, and if you can avoid losing beads at every one of them, you maximize your library yield. If you have any additional technical questions, you can reach us at [email protected]
So, Andrew, thanks for joining us today.
My pleasure, thanks Deana.
And as always, if you have any suggestions for future episodes, please let us know.
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