Fragmented Nuclear DNA Is the Predominant Genetic Material in Human Hair Shafts

Abstract

While shed hairs are one of the most commonly encountered evidence types, they are among the most limited in terms of DNA quantity and quality. As a result, nuclear DNA short tandem repeat (STR) profiling is generally unsuccessful and DNA testing of shed hair is instead performed by targeting the mitochondrial DNA control region. Although the high copy number of mitochondrial DNA relative to nuclear DNA routinely permits the recovery of mitochondrial DNA (mtDNA) data in these cases, mtDNA profiles do not offer the discriminatory power of nuclear DNA profiles. In order to better understand the total content and degradation state of DNA in single shed hairs and assess the feasibility of recovering highly discriminatory nuclear DNA data from this common evidence type, high throughput shotgun sequencing was performed on both recently collected and aged (approximately 50-year-old) hair samples. The data reflect trends that have been demonstrated previously with other technologies, namely that mtDNA quantity and quality decrease along the length of the hair shaft. In addition, the shotgun data reveal that nuclear DNA is present in shed hair and surprisingly abundant relative to mitochondrial DNA, even in the most distal fragments. Nuclear DNA comprised, at minimum, 88% of the total human reads in any given sample, and generally more than 95%. Here, we characterize both the nuclear and mitochondrial DNA content of shed hairs and discuss the implications of these data for forensic investigations.

Evidence for multi-copy Mega-NUMTsin the human genome

“…Tissues that naturally lack intact nDNA, such as single thrombocytes, exclusively yielded the V mitotype. Hair shafts that are known to contain only minute amounts of high molecular weight nDNA ( 61 ), also yielded only the V mitotype in 55 of 56 analyzed hair shafts. Only one of the seven hair shaft samples from individual IV.5 showed the U mitotype in addition to the V mitotype.…”

“…However, we suggest the sequence analysis of hair shafts from the affected individual as an elegant alternative. While hair shafts usually contain sufficient amounts of mtDNA for PCR-based analyses, they typically contain only minute amounts of highly degraded nuclear DNA ( 61 ). Consequently, PCR assays aiming at mtDNA target sequences will almost exclusively yield amplicons deriving from the actual mtDNA copies being present in a hair shaft, as demonstrated in our study.…”

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

“…sequencing error, PCR error, DNA damage, pseudogenes) could be consistently distinguished from authentic signal. We have employed methods for doing exactly that in other NGS studies [23,56,57]. However, these analyses have been largely manual, which is not suitable to a production environment.…”

“…Hair samples were extracted according to Ref [23], while bone samples were extracted using a variation of the protocol described in Ref [24]. Extracts were quantified using either the Quantifiler Duo assay (Thermo Fisher Scientific, Waltham, MA), an internally validated mtDNA-specific qPCR assay [25], or both.…”

Interpol review of forensic biology and forensic DNA typing 2016-2019

“…A special issue on forensic genomics was organized in Genes [ 217 ] by guest editors Manfred Kayser and Walther Parson with 11 articles published between November 2017 and December 2018 (see https://www.mdpi.com/journal/genes/special_issues/Forensic_Genomics ). Topics for these open access articles include performing molecular analysis of the RNA transcriptome for human organ tissue identification to assist in investigations of traumatic injury [ 218 ], investigating the epigenetic discrimination of identical twins using reference sample buccal swabs and saliva and cigarette butts common to forensic evidence [ 163 ], recovering fragmented nuclear DNA from human hair shafts [ 219 ], using high-throughput sequencing to recover nuclear DNA from a 4000-year-old Egyptian mummy head [ 220 ], examining mitochondrial DNA heteroplasmy with MPS to help distinguish maternal relatives [ 221 ], dating juvenile blow flies to assist forensic entomology with postmortem interval estimation [ 222 ], predicting the postmortem interval using microbiome data [ 223 ], applying NGS probe capture enrichment techniques to examine the whole mitochondrial genome and 426 nuclear SNPs on individual telogen hairs [ 224 ], and demonstrating that flanking region variation can impact rates of stutter product formation in STR markers [ 225 ].…”