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Hi Everyone, my name is Efi Melista and I’m the Head of Field and Lab Operations at Omixon.
And today we’ll be talking about what is new in our brand new Holotype HLA v.2 protocol.
A lot of you are already familiar with the original protocol – the “V.1” as we call it -
and we are happy to present a faster and simpler version of that protocol
to make HLA typing by NGS faster and easier for everybody.
The turnaround time from genomic DNA to genotype is less than 48 hours for 24 samples at any
number of loci that you are interested when sequenced on a micro flow cell.
It’s very important to know that the hands-on time for 24 samples is less than 4 hours with
this new version of the protocol, so it’s quite nice, it’s quite convenient and "lab tech friendly".
As usual, remember that we have several safe stopping points, jut like in the V.1 protocol,
so you can essentially stop the whole procedure and freeze your samples at the end of every single step.
Store them at -20 degrees and come back to continue the process when you’re ready for it.
The one thing that I would like to point out is that part of the whole process is not technically
part of the protocol timings, such as the DNA extraction, which is expected to be different and separate.
So let’s begin with the highlights and differences of this V.2 protocol.
As usual, these are the steps that describe the protocol, and let’s start from the beginning.
We have removed the Agarose Gel Verification after the PCR amplification, so you don’t
have to run gels anymore.
The amplicon quantitation provides exactly the same information to tell you if an amplification
has worked or not.
We have also simplified the amplicon quantitation step to make it a bit more streamlined, to
reduce the amount of pipetting, and just to make the step a little bit faster and easier for everyone.
At the end of step 3 essentially of the whole process, where we have the amplicon quantitation
normalization and pooling, we have changed the standard ExoSAP enzyme for the PCR purification
with the ExoSAP Express, which is essentially the same kind of enzyme: it works the same way, but it’s faster.
So instead of spending one hour during purifying your PCR products, now you spend five minutes
in the PCR.
Very convenient, very fast.
The next big difference is that for the library quantitation (in step 6), you can exchange
using the qPCR machine and the Kapa Biosystems kit with using the Qubit and also the Qubit
reagents, the broad-range assay kit, which saves you about 45 minutes of the whole process.
Finally another major difference that reduces quite a bit of time is the sequencing on the
Illumina MiSeq, which now we support sequencing your library with a 300-cycle chemistry and
any flow cell essentially that fits the number of samples and loci that you’re processing.
So as a reminder: 24 samples at 11 loci can easily fit in a micro flow cell, which takes
about 17 hours.
So essentially you could be starting the whole process in the afternoon of Day 1 beginning
with the long-range PCR amplification, you could be loading the MiSeq before the end
of Day 2, which means that the MiSeq sequencing reactions will be finishing by mid-day on
Day 3, and you can be analysing and reviewing results by the end of Day 3.
So while before, in the V.1 protocol, the turnaround time was closer to 5 days,
the turnaround time with the new protocol has been reduced down to less than 3 days including
sequencing and analysis review, which is quite convenient.
One last thing that I want to point out is that exactly because we have streamlined the
protocol even more, every single step of the process can be very easily automated on various
and on different number of liquid handlers – anything from low-budget to higher budget,
higher capacity to lower capacity - pretty much anything that would fit your own workflow.
Feel free to contact us for a lot more details, not only on the automation, but even more
so on the Holotype HLA V.2 protocol.
Feel free to contact us directly at sales@omixon.com or at support@omixon.com for email correspondence,
or call us – you all know our numbers and who we are anyway.
Thank you for joining and see you again next time!
Bye!