In the open discussion webinar hosted by Frontline Genomics, Joshua Burgess Ph.D., Manager Scientific Research, R&D at Illumina and Rafal Iwasiow, Ph.D., Vice President, R&D at DNA Genotek explain how the Nextera DNA Flex and Oragene DNA from saliva kits are complementary technologies shifting the paradigm by enabling direct saliva input in the creation of high-quality libraries for whole-genome sequencing (WGS).

In the first half of the presentation, Joshua Burgess explains how the Nextera DNA Flex (Illumina; San Diego, CA) represents the next era in library prep and has a lot of advantages over traditional library prep methods for WGS. The foundational technology is the bead-linked transposome that saturates at 100 ng, so any additional sample input falls off. This means that you do not need the same level of rigor in quantification, and still have a normalized sample integration at the output. This technology is flexible enough to support human WGS with ultra-low DNA input samples, and is supportive for bacterial WGS and metagenomics. The real advantage to this kit, however, is with the direct sample input, because you can go from a raw sample, such as saliva, to a library in 3.5 hours, and get consistent data from sample to sample. This is a very novice-friendly protocol.

“We know that we have a fast protocol because if you use direct saliva input, you can start in the morning and have your libraries by lunch in three and a half hours. We also know that data is just as good with genomic DNA as it is with the raw samples.” (Joshua Burgess Ph.D., Illumina)

In a collaborative study by Illumina with DNA Genotek, the data showed Oragene and Nextera DNA Flex are compatible and offer a solution to direct-to-assay WGS. Illumina collected fourteen saliva samples in Oragene self-collection kits (DNA Genotek; Kanata, ON) stored at ambient temperature. Thirty (30) µl by volume of Oragene/saliva raw sample was added directly into the Nextera DNA Flex workflow, completely skipping a quantification step. All samples were super-saturated exceeding the 100 ng amount, with actual DNA quantities ranging from 174 ng-2084 ng in the 30 µl aliquot. Libraries of similar yields were successfully produced with 13% CV, ready for WGS.

“All this data was developed with help and collaboration from DNA Genotek. The Oragene tubes are a fantastic technology and they’ve been absolutely critical to us … in developing this particular workflow and getting the high quality data we’ve been able to retrieve.” (Joshua Burgess Ph.D., Illumina)

In the latter half of the presentation, Dr. Iwasiow focused on the attributes of Oragene that make saliva a particularly well-suited source of DNA in sequencing studies. The main source of DNA in saliva is the same as that in blood, with ~70% of DNA coming from white blood cells. With Oragene kits, the extracted DNA is very high quality and quantity with on average median yield of 48 µg per 2 ml saliva sample. Oragene/saliva samples are stable at ambient temperatures and are a painless, non-invasive, and easy collection method that dramatically increases study compliance. Since saliva is considered an exempt human specimen, it’s a cost-effective means of shipping a sample in the post without any need for cold chain logistics. Finally, and importantly, the bacterial content in Oragene-collected saliva is very low (on average ~11%) and this bacteria is neutralized once collected in Oragene. This means any bacteria present in a saliva sample does not grow or degrade the human DNA sample.

Key take-aways:

• Nextera DNA Flex provides a fast simple workflow solution for most WGS applications;
• Using Oragene, libraries can be generated from direct saliva input in 3.5 hours;
• Saliva workflow is optimized to provide normalized output;
• Automation scripts for saliva-based Nextera DNA Flex sample preps exist for most platforms;
• Sequencing metrics from Nextera DNA Flex is on-par with existing mechanical-based fragmentation methods;
• Collections with Oragene saliva kits will increase donor compliance and reduce the costs and complexities associated with DNA collection, transport and storage.

Finally, here are a few snippets from the Q&A period at the end:

(Frontline Genomics Host) “Integrating or introducing NGS is an order of magnitude harder than anything else that’s come before it. I think now we’re at a point where sequencing technology has gotten that much better – that much cheaper – that much quicker – but there’s still a relatively high barrier to entry just from a skills perspective when you look at all the stuff that happens both upstream and downstream of the sequencers themselves. How is the industry addressing some these bottlenecks on either side of the sequencing itself?”

(Joshua Burgess, Illumina) “I think historically some of these library prep methodologies have been a little more challenging – a bit more finicky – they require the time and the training to make sure you can get the high quality libraries that you need. But with Nextera DNA Flex what we really try to hammer on is making sure that it is simple. That a person can address this and get the high quality results every single time. They can count on those results regardless of their input – regardless of their sample type. Of course there is always a big emphasis on trying to make this as cost effective as possible for the customer because we know that’s a bottleneck and it’s certainly an area of focus.”

(Frontline Genomics Host) “Similar question, looking at the stuff DNA Genotek is doing – you’re making things a lot easier as well. What’s the drive behind the R&D that you guys do?”

(Rafal Iwasiow, DNA Genotek) “We focus a lot on quality of sample. That’s where we start – but we want to make that high quality sample really accessible and easy to obtain. In the context of what we’re talking about today, it means that you can collect a lot of samples from wherever you want and get them to the lab and process them with real simplicity.”

(Frontline Genomics Host) “Are there any particular areas of research or applications that you think this is going to make easier, and open up new doors as well?”

(Joshua Burgess, Illumina) “The hope is that with the way we developed Nextera DNA Flex the applications should be pretty close to limitless. If you can get genomic DNA, we like to think you can probably get some high quality data from those libraries. With direct sample input we continue to try and expand in those areas as much as we can. We have the new application in metagenomics we’re very excited about as well. We’re always interested in expanding our applications and we’re always trying to find new areas to prove out Nextera DNA Flex can perform in those areas – and we believe that will enable our customers in a plethora of different areas.”

(Rafal Iwasiow, DNA Genotek) “From our perspective, as sequencing technology enters a lot more labs, we’re looking at simplifying the process. And I think both the non-invasive Oragene collection and the Nextera Flex really hit those two things.”

(Frontline Genomics Host) “Is it possible to use libraries made by Nextera DNA Flex for downstream probe-based capture? In other words, can we use this for targeted sequencing on a panel of genes?”

(Joshua Burgess, Illumina) “I think this is an important point to get across: this particular kit is optimized specifically for whole-genome sequencing. We do not recommend using any type of capture-based methods with Nextera DNA Flex.”

Listen to full version by watching the webinar re-play at your convenience. As always, we look forward to hearing your thoughts and opinions in the spaces below.

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