The impact of common PCR inhibitors on forensic MPS analysis

Research output: Contribution to journalArticle

Abstract

Massively parallel sequencing holds great promise for new possibilities in the field of forensic genetics, enabling simultaneous analysis of multiple markers as well as offering enhanced short tandem repeat allele resolution. A challenge in forensic DNA analysis is that the samples often contain low amounts of DNA in a background that may interfere with downstream analysis. PCR inhibition mechanisms of some relevant molecules have been studied applying e.g. real-time PCR and digital PCR. However, a detailed understanding of the effects of inhibitory molecules on forensic MPS, including mechanisms and ways to relieve inhibition, is missing. In this study, the effects of two well-characterized PCR inhibitors, humic acid and hematin, have been studied using the ForenSeq DNA Signature Prep kit. Humic acid and hematin resulted in lowered read numbers as well as specific negative effects on certain markers. Quality control of libraries with Fragment analyzer showed that increasing amounts of inhibitors caused a lowered amplicon quantity and that the larger amplicons were more likely to drop out. Further, the inhibitor tolerance could be improved 5–10 times by addition of bovine serum albumin in the initial PCR. On the contrary to the samples with inhibitors, low-template samples resulted in lowered read numbers for all markers. This difference strengthened the conclusion that the inhibitors have a negative effect on the DNA polymerase activity in the initial PCR. Additionally, a common capillary gel electrophoresis-based STR kit was shown to handle at least 200 times more inhibitors than the ForenSeq DNA Signature Prep kit. This suggests that there is room for improvement of the PCR components to ensure analytical success for challenging samples, which is needed for a broad application of MPS for forensic STR analysis.

Details

Authors
Organisations
External organisations
  • Swedish National Forensic Center
  • National Institute of Standards and Technology
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Forensic Science

Keywords

  • ForenSeq DNA signature prep kit, Hematin, Humic acid, Massively parallel sequencing, MiSeq FGx, PCR inhibitors, Short tandem repeats
Original languageEnglish
Pages (from-to)182-191
Number of pages10
JournalForensic Science International: Genetics
Volume40
Publication statusPublished - 2019
Publication categoryResearch
Peer-reviewedYes

Related research output

Maja Sidstedt, 2019 May 24, Lund University, Division of Applied Microbiology: Department of Chemistry, Lund University. 172 p.

Research output: ThesisDoctoral Thesis (compilation)

View all (1)