Difficult and highly degraded DNA samples are the bane of every forensic DNA analyst. When possible, DNA samples are collected, stored and transported from the crime scene to the laboratory carefully and with the proper protocols in place.
Of course, analysts can’t always be that lucky. Touch evidence, hairs and skeletal remains that have been exposed to the elements push the limits of current forensic DNA typing technologies. In a new proof-of-concept study, researchers at the University of North Texas (UNT) have developed, in collaboration with NimaGen, a reverse complement PCR (RC-PCR) short amplicon 85 SNP-plex panel that can be used to improve library preparation for DNA analysis of these difficult samples.
If DNA has been severely degraded to lengths as short as 50 bases in length, PCR amplification becomes highly challenging, and the size of the fragments makes it nearly impossible to detect DNA markers. The study authors say a more successful approach to typing highly fragmented DNA comprises the reduction of amplicon sizes of short tandem repeats (STRs) or single nucleotide polymorphisms (SNPs).
RC-PCR is a novel target enrichment and library preparation method for next generation sequencing. The method uses two reverse-complement, target-specific primer probes as well as a universal primer that hybridize to generate target-specific index primers that are then capable of multiplex amplification of target regions. Since target enrichment and indexing are performed in a single closed-tube system, the number of sample handling steps is reduced, which also substantially reduces turnaround time and contamination risk.
“These features of RC-PCR make the technology a unique application to successfully target SNPs in fragmented and low copy number DNA and yield results from samples in which no or limited data are obtained with standard DNA typing methods,” the authors write in their paper, published in BioTechniques.
The team’s first attempt to design a panel with 50 bp-long SNP amplicons resulted in only 27 markers meeting the criteria. On mock forensic samples as well as challenging casework bone samples, the method revealed high sensitivity, successful analysis in the presence of PCR inhibitors and even the potential to detect mixed DNA profiles.
Still, the forensic scientists knew they had to increase the number of SNPs if the panel had hopes of real-world forensic applications. They did so by expanding the amplicon length to approximately 100 bp— add 60 SNPs to ultimately produce a multiplex panel of 85 identity SNPs.
Preliminary tests of the RC-PCR 85-plex demonstrated sensitivity and concordance, according to the study results. The percent of recovered SNP loci (showing at least one allele) ranged from 78% at the lowest DNA input of 31 pg to over 99% at 62 pg. Allelle dropout was seen at 8% and 6%, respectively.
“The data obtained for low DNA inputs ≤125 pg are promising since highly degraded samples tend to be of low copy number; even partial SNP profiles can provide critical information for missing persons and criminal cases,” write the authors.
While this proof-of-concept study shows support for the RC-PCR system on forensic samples, the authors say the newly developed panel should be tested on challenging DNA samples in future studies.
“The next steps of testing will include analysis of the IDseek SNP85 panel in different human populations, mixture detection, inhibition resistance and performance on degraded forensic samples and human remains,” the authors conclude. “The technology may also be beneficial for typing formalin-fixed paraffin-embedded tissue samples. Given the diversity and throughput of [next generation sequencing], other types of SNPs (e.g., ancestry informative SNPs) and other markers (e.g., short STRs and mitochondrial DNA) can be incorporated into the same multiplex system.”