Next Generation Sequencing of Highly Damaged, Degraded DNA

A critical problem in preparing libraries for Next-Generation Sequencing (NGS) stems from the quality of input DNA.  Samples are often damaged or degraded, resulting in failed, poor quality, or highly biased libraries using traditional methods.  Further, the challenge of accurately representing AT- and GC-rich regions of the genome can be exacerbated when the DNA is damaged or degraded.

We have developed two library preparation methods to generate libraries that accurately reflect the original DNA input, even when that input is damaged or degraded.  The Accel-NGS 2S family of DNA library kits adapts double-stranded DNA molecules with high efficiency, and the Accel-NGS 1S Plus DNA library kit adapts single-stranded DNA molecules in a template-independent manner.

As a pilot study, we obtained vaquita DNA from NOAA Fisheries’ Southwest Fisheries Science Center.  The vaquita is the world’s most endangered marine mammal and the tissue available is highly degraded due to post-mortem sampling and sub-optimal long-term preservation and storage conditions.  Using the Accel-NGS 1S Plus DNA library kit, libraries were generated with zero sample failures and an average nuclear DNA alignment rate of 93%.  Additionally, the Accel-NGS 2S PCR-Free kit was used to construct libraries from degraded Harbor porpoise samples with an alignment rate of 96%. 

We have demonstrated the ability of the Accel-NGS 1S and 2S kits to generate libraries with minimal bias from highly degraded porpoise samples.  With these kits, scientists are empowered to sequence DNA previously thought to be unusable for NGS due to their damaged or degraded nature.