A Simple Workflow to Confirm Your NGS Variants – Seq It out #17

Previously, I talked about why you should confirm your NGS or Next Gen Sequencing variants with Sanger sequencing.  Today let me give you some quick tips and tricks on how to make this process a snap!

So you are seeing some variants in your NGS data that you are not confident about. How can you quickly re-sequence a particular locus of interest?

Let’s take a look at the workflow

First thing to do is to find the correct primers for your resequencing reactions and this has become an easy task thanks to the Primer Designer Tool which we reviewed in a previous Seq It Out segment

If you are using an Ion Torrent system, you can access this tool easily through Ion Reporter.   Just check your variants of interest and select ‘Order CE primers’ from the top button.

For other NGS platform users, you can access the tool through our website: thermofisher.com/primerdesigner. You will need to prepare a shortened .vcf file containing just the variants to be verified and through the tool, you can find primer pairs for any human exome and mitochondrial genome.

Next is performing the Sanger sequencing reaction.  If you want to cut some time, the BigDye Direct Sequencing Kit can save you approximately 3 hours compared to the traditional Sanger sequencing workflow.  The new workflow with BigDye Direct combines the PCR clean-up and cycle sequencing into a single step and requires a lot less hands-on time. However, using this workflow does require your primers to be ordered with M-13 tails, which is also available through the Primer Designer Tool.

Finally, we need to compare the Sanger sequencing result with the NGS result. This can be easily done with using the NEW Next Generation Sequencing Confirmation (NGC) module on the Thermo Fisher Cloud. Just upload your NGS .vcf file, your sanger sequencing traces, and the assay id numbers for your PCR primers if you used the Primer Designer Tool.

A few clicks and you will get an alignment of NGS variant and Sanger sequence traces with the human genome where you can visually verify the variants.  You can also generate a comprehensive report or a joint NGS/Sanger .vcf file for further downstream bioinformatics analysis.

You see, NGS Confirmation with Sanger Sequencing is no witchcraft. Everybody can do it.

If you have more questions on NGS confirmation, just submit them at thermofisher.com/ask and subscribe to our channel to see more videos like this.

Remember, when in doubt just Seq It Out !