You’ve probably heard about the new Minor Variant Finder software, and how it will enable 5% somatic variant detection using Sanger sequencing. Give me 5 minutes today, and I will show you how the software works step by step.
Minor Variant Finder is perfect for those requiring a cheap, fast, and accurate way to call low frequency variants when working with a small number of targets. It is also a great tool for doing NGS confirmation with Sanger sequencing. So how do we get Minor Variant Finder in your lab?
First, visit www.thermofisher.com/mvf. Here, you can download a demo version or purchase the full version. The software installs locally on your computer, and runs in your web browser. Once installation is completed, just click on the icon and your web browser will open automatically. Minor Variant Finder will greet you with all your active projects, allowing you to manage them in one spot. Let’s start a new project.
Here we see the traces view and this is where you import your trace files. After uploading your ab1 files, the software will give you an overview of the quality of your files. Green represents a good quality score and red means a bad score. Double click on the file name and take a closer look at the traces. The quality score is determined by a set of parameters, and you have the option to define those parameters such as signal to noise, trace score, read length and so forth.
This data looks pretty good, so let’s move on to the reference tab. There are two ways to create a reference; one is to use the normal control as a reference, which is the easiest way. The second is to create a reference and/ or upload an NGS file.
We will use the control for reference here, so let’s just move to the organize tab.
The software compares the forward and reverse traces of your control sample to the forward and reverse traces of your test samples. You can drag your files manually into the control and the test specimen buckets. So first, create an amplicon. Move the controls to the control buckets. Then create a folder for each test specimen.
Another option is the auto-organize function using a naming convention. Click on organize, and tell the software which part of the file name is the amplicon, the specimen, and the control. Click organize, and everything is auto-organized.
Next, go to the Analysis tab, verify that all your data is set up correctly and click on Run Analysis.
And here are your results. Here are the reference traces and also the minor variants identified in the test sample files. If you go to the table view, you can see a list of the candidate minor variants, with percentages in both forward and reverse strands. The color here is a review indicator that tells you how much attention you should pay when inspecting these variants. Green means that algorithm has high confidence that it made the right call. Yellow means it should be followed by a visual inspection of the electropherogram. And red means that algorithm is not quite sure, so very critical inspection is needed.
Let’s examine these traces by looking at the electropherogram. Here are the Forward traces and here are reverse traces. This is the control, test sample, and our noise minimized results. The minor variant called is here. Use the slider to adjust the electropherogram for a closer inspection. The percentage of both the forward and reverse are on top. And it should be similar for same variant. If all data convinces you that this is a true variant, you can click accept. If you disagree, you can insert a comment. And that’s it for review.
Click on report, and you can generate a variant file of all your findings.
Minor Variant Finder is also a great tool to confirm your NGS results. Check out the user guide on detailed instructions on how to set up a reference, a NGS vcf file for comparison, and how to get a combined report.
And download your copy of Minor Variant Finder at http://www.thermofisher.com/mvf
Access the full user guide at the link below: http://tools.thermofisher.com/content/sfs/manuals/MAN0014835_MinorVariantFinder_v1_UG.pdf?cid=techcomm91c