Learn How DNA Integrity Affects Detection Sensitivity and What You Can Do to Maximize Sensitivity

By Laura Griffin, Ph.D. on Fri Feb 5, 2016


Summary

  • DNA damage interferes with PCR during library preparation, so variants present on damaged fragments are not represented in sequencing library.
  • Variant detection sensitivity is therefore driven by the integrity of input genomic DNA.
  • Increasing the input of genomic DNA for library preparation improves detection sensitivity.
  • To maximize detection sensitivity, measure DNA integrity with the Archer™ PreSeq™ DNA QC Assay and calculate the appropriate amount of input DNA for library preparation with Archer’s online amplifiable genomes calculator.

Next-generation sequencing (NGS) is an invaluable tool for discovering and detecting genomic variants, especially when performed with target-enriched DNA libraries, such as those generated by Anchored Multiplex PCR (AMP™). AMP-based NGS enables increased read depth of target sequences, thereby enhancing the sensitivity of variant detection in those genomic regions. This is particularly useful in detecting rare variants that would otherwise be undetectable by whole-genome NGS assays, such as those with low allele frequencies or those in a heterogeneous population (i.e., samples with low tumor cellularity). Yet even with this AMP-enabled enhancement of detection sensitivity, researchers should consider other factors that can limit sensitivity. So what are these factors? And what can the researcher do to address them and ensure optimum detection sensitivity?

Typically, poor genomic DNA integrity resulting in poor sequencing coverage is the culprit in limiting the sensitivity of variant detection. Routine formalin-fixed paraffin-embedded (FFPE) storage of clinical specimens severely damages DNA, inducing DNA fragmentation, cross-linking of proteins to nucleic acids, strand cleavage, and base modifications. Although AMP can capture degraded genomic fragments too small to be amplified by opposing primer-based techniques, DNA damage within a fragment can still interfere with PCR and thus library preparation. These alterations can result in a low complexity library generated by amplification of only a portion of the genomic fragments originally present in the sample. If a variant is present on a region of the genome that is not amplified efficiently, it won’t be represented in the library and it won’t be detected by downstream NGS assays. As the Archer™ VariantPlex™ panels demonstrate a mean coverage uniformity of >96%, detection sensitivity is primarily driven by the integrity of the input genomic DNA. Consider the examples illustrated below.

Anchored multiplex PCR (AMP) with undamaged genomic DNA, where all fragments are amplifiable and thus are all represented in sequencing library for NGS, with high library complexity and sensitivity of variant detection.

Here, perfectly intact, undamaged genomic DNA is 100% amplifiable for AMP-based library preparation. Out of 5 genomic fragments originally present, all 5 are amplifiable by GSP1/2 and thus all 5 are represented in the sequencing-ready library, maximizing detection sensitivity. In theory this is great, but what would happen to the library if the sample were severely damaged?

Anchored multiplex PCR (AMP) with degraded genomic DNA sample, such as FFPE specimen, where only some fragments are amplifiable and represented in the sequencing library for NGS, resulting in a low complexity library and poor coverage and detection sensitivity.

DNA damage between the GSP1/2 binding sites and the cleavage site/start site, including fragmentation or crosslinking, interferes with nested PCR during library preparation. Out of 5 genomic fragments originally present, only 2 are amplifiable and thus only these 2 are represented in the sequencing-ready library. Therefore…

Increasing the input of amplifiable genomes for AMP-based library preparation results in a more complex libary with more unique start sites per gene-specific primer (GSP) and increased sensitivity of variant detection in NGS assays.

By this logic, researchers can improve detection sensitivity by increasing the input of amplifiable genomes for AMP-based library preparation prior to NGS assays. The number of amplifiable genomes can in fact be calculated based on the DNA QC score obtained from the Archer™ PreSeq™ DNA QC Assay. This assay uses qPCR to amplify a 100bp sequence to estimate the number of amplifiable genomes relative to a reference sample. It is important to note that the absolute mass of DNA in a sample does not correlate to the number of amplifiable genomes across samples, as the ability of a fragment to be amplified depends on the integrity of DNA, which varies between samples. To simplify matters, Archer’s scientists have determined the minimum and optimum numbers of amplifiable genomes required for various downstream applications. Archer’s new online genome calculator calculates the minimum and recommended DNA input amounts for these applications based on the DNA QC score.

So what can you do to ensure optimum variant detection sensitivity?

  1. Check the integrity of your DNA using the Archer™ PreSeq™ DNA QC Assay
  2. Prepare libraries using the recommended amount of DNA input from the online amplifiable genomes calculator
  3. Confirm your library’s complexity by checking the mean unique start sites per GSP2 in Archer Analysis
  4. Further increase DNA input if necessary

For a more comprehensive review of factors influencing detection sensitivity and the PreSeq DNA QC Assay design and implementation, see our Technical Note, “Beyond Nanograms: Amplifiable Genomes to assess NGS library complexity from FFPE samples.”



About Laura Griffin, Ph.D.

Laura Griffin earned her PhD in Microbiology from the University of Colorado Denver, Anschutz Medical Campus. Her research focused on cancer virology, dissecting virus-host interactions during Human Papillomavirus infection. Laura is passionate about cancer research as well as effective education and communication of scientific ideas. Laura joined the ArcherDX team as Scientific Editor in January, 2016. In her spare time, Laura is a group fitness instructor and enjoys cycling, hiking, skiing and snowshoeing in the Rocky Mountains of Colorado.

How to contact us

Address

2477 55th Street, Suite 202

Boulder, CO 80301

Phone

Phone: (877) 771 1093

Phone: (303) 357 9001

All content © 2017 ArcherDX, Inc.

For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.