There are lots of reasons to love a PCR-free library for next-generation sequencing (NGS) workflows: avoiding the introduction of bias from an amplification step, shortening workflow times, and eliminating errors and duplications caused by PCR. But these useful libraries can flummox standard quality control protocols, making it harder for scientists to have confidence in the downstream results of their sequencing experiments.
At Quantabio, we are all about eliminating variables and ensuring a consistent, high-performance experimental processes. We want to make it easier for scientists to enjoy the advantages of PCR-free libraries without any confusion or frustration.
That’s why we have developed resources to improve the quality control process for PCR-free library workflows. In a new application note, we describe a method to accurately determine the size of PCR-free libraries by adding a short PCR amplification step prior to automated DNA fragment analysis. This overcomes the well-known problem that standard QC of these libraries can mistakenly make it appear that DNA strands are much longer than they actually are. Check out the application note for specific methods and illustrated results of PCR-free library QC with and without this key step.
We also created a recent webinar to go over this topic, and the on-demand recording is now available. Speakers include Patrick Schindele from Agilent Technologies and Daniel Teutsch from Quantabio. Clocking in at about 30 minutes, the webinar is a great educational tool you can watch while eating lunch. But if you don’t have time, here are a few highlights:
Garbage in, garbage out: The Agilent TapeStation can be used at multiple points in the sample prep and library prep processes to ensure the quality of the DNA that will be used for an experiment for optimal results. Working with low-quality samples risks wasting time, sample, and lab resources that could have been better used elsewhere.
Migration patterns: PCR-free libraries migrate differently in gel capillaries due to their Y-shaped adapters, making it look like DNA is larger than it really is. Performing PCR on certain primers will create linearized libraries that will migrate as expected through the gel, allowing scientists to test a small amount of their library and confirm that sizing is what’s desired.
Quantabio kits to the rescue: Our sparQ DNA Frag & Library Prep Kit and sparQ DNA Library Prep Kit support the generation of PCR-free libraries with minimal hands-on time. These kits enable tunable and reproducible enzymatic fragmentation, meeting the gold standard of shearing, while delivering excellent yield.
Questions about QC for NGS workflow library prep? Don’t hesitate to ask us.