Why Confirm Your NGS Variants – Seq It Out #14

Today’s topic is the confirmation of next gen sequencing or NGS results using good old Sanger sequencing. You may ask “why on earth would I want to do that?  This sounds really backwards!” Well, just like a famous saying from an American President, Trust – but verify.

Did I just say you may want to verify your NGS data?

Don’t get me wrong here. Next-Gen sequencing has evolved over the last couple of years to become a robust technology regardless of the application that it is used for.

Researchers like you probably attempt to identify the role that certain variants may play in disease susceptibility, disease progression, or phenotypic variation. So identifying accurate sequence variants is critical for your research result.

Let’s take a look in our lab book on why you may want to verify.

The chemistries used in NGS technologies enable highly sensitivity detection of low level variants; however, this may sometimes give rise to low levels of sequencing errors that may lead to confusing or incorrect interpretations.

A source of uncertainty can arise from uneven coverage of NGS reads; this can happen because not all sequences can be sequenced with the same efficiency. Sequences that are more difficult for the polymerase to read through due to secondary structure, highly repetitive sequences, or other factors, will be underrepresented in the subsequent data files.

In addition, some NGS targeted sequencing approaches depend on capture by hybridization before library preparation. This hybridization can be highly sequence-dependent and can affect the representation of some sequences in the final libraries. And because confidence in the results is highly dependent on the number of instances that sequences are detected, any regions that are underrepresented in the final data output could be more suspect, making you wonder if those variants are real.

There could also be sequencing biases that are specific to the platform being used. For example, GCrich regions can be particularly hard to read through. Therefore, they can introduce misincorporation errors, the specific type of which can be influenced regardless of the sequencing instrument being used.  Similarly, errors can be introduced when sequencing through long homopolymer sequences. Thankfully, with the latest advances in Ion Torrent sequencing technologies, this has been less of an issue.

And again this applies to all NGS systems.

So now you understand the reasons to confirm NGS variants, let’s talk about why Sanger sequencing is the best method to use for confirmation.   If you’ve been in the sequencing world for a while, you are probably already familiar with this simple workflow.  Sanger sequencing allows you to sequence one single strand at a time with 99.99% accuracy.  It is extremely reliable as a verification tool and still considered the gold standard. And for the small amount of samples that require confirmation, Sanger Sequencing is the most cost effective sequencing method.

I hope this video was helpful regarding NGS confirmation, but I am sure you’ll have more questions.

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And remember, when in doubt, just Seq It Out