Extended kinship analysis of historical remains using SNP capture.

DNA-assisted identification of historical remains requires the genetic analysis of highly degraded DNA, along with a comparison to DNA from known relatives. This can be achieved by targeting single nucleotide polymorphisms (SNPs) using a hybridization capture and next-generation sequencing approach suitable for degraded skeletal samples. In the present study, two SNP capture panels were designed to target ~ 25,000 (25 K) and ~ 95,000 (95 K) nuclear SNPs, respectively, to enable distant kinship estimation (up to 4th degree relatives). Low-coverage SNP data were successfully recovered from 14 skeletal elements 75 years postmortem using an Illumina MiSeq benchtop sequencer. All samples contained degraded DNA but were of varying quality with mean fragment lengths ranging from 32 bp to 170 bp across the 14 samples. SNP comparison with DNA from known family references was performed in the Parabon Fx Forensic Analysis Platform, which utilizes a likelihood approach for kinship prediction that was optimized for low-coverage sequencing data with cytosine deamination. The 25 K panel produced 15,000 SNPs on average, which allowed for accurate kinship prediction with strong statistical support in 16 of the 21 pairwise comparisons. The 95 K panel increased the average SNPs to 42,000 and resulted in an additional accurate kinship prediction with strong statistical support (17 of 21 pairwise comparisons). This study demonstrates that SNP capture combined with massively parallel sequencing on a benchtop platform can yield sufficient SNP recovery from compromised samples, enabling accurate, extended kinship predictions.

Capture enrichment and massively parallel sequencing for human identification.

In the past decade, hybridization capture has gained attention within the forensic field for its possible use in human identification. One of the primary benefits to capture enrichment is its applicability to degraded DNA fragments that, due to their reduced size, are not amenable to traditional PCR enrichment techniques. Hybridization capture is typically introduced after genomic library preparation of extracted DNA templates for the subsequent enrichment of mitochondrial DNA or single nucleotide polymorphisms within the nuclear genome. The enriched molecules are then subjected to massively parallel sequencing (MPS) for sensitive and high-throughput DNA sequence generation. Bioinformatic analysis of capture product removes PCR duplicates that were introduced during the laboratory workflow in order to characterize the original DNA template molecules. In the case of aged and degraded skeletal remains, the fraction of endogenous human DNA may be very low; therefore low-coverage sequence analysis may be required. This review contains an overview of current capture methodologies and the primary literature on hybridization capture as evaluated for forensic applications.

Next generation sequencing of STR artifacts produced from historical bone samples.

STR artifacts are commonly observed in electrophoretic data and can complicate interpretation of the profiles produced. Even when a consensus approach is applied, reproducible artifacts have the potential to convolute the analysis. DNA obtained from historical bone samples is often heavily degraded and damaged, requiring the use of more sensitive procedures to increase allele recovery. Additionally, skeletal remains exposed to environmental conditions may be afflicted with microbial DNA contamination that cross-reacts with the primers during short tandem repeat (STR) multiplex amplification. STR artifacts manifested as a result of these circumstances can be sourced and characterized using new sequencing technologies to potentially ease the analysis burden. For this study, PCR product from 17 low-quality bone samples exhibiting reproducible autosomal and Y-chromosomal STR (Y-STR) artifacts in capillary electrophoresis (CE) data were sequenced with next generation sequencing (NGS). Sequenced reads were bioinformatically sorted using STRait Razor to determine the authenticity of alleles and confirm the profile generated by CE. Sequence data from the PCR products and a subset of the associated extracts were further analyzed with Kaiju to classify the microbial species present and identify potential sources of artifact peaks. A suspected Y-STR artifact was similar in sequence to Pseudomonas sp. BAY1663, a species ubiquitously found in soil. Regions of homology were observed between the Pseudomonas genome and the presumed primer binding locations for Y-STRs included in the AmpFlSTR Y-Filer STR kit. Characterization of such supposed artifact peaks may aid in interpretation of CE data and ultimately lead to increased confidence in the reported results.

A Forensic Genomics Approach for the Identification of Sister Marija Crucifiksa Kozulic.

Sister Marija Krucifiksa Kozulic (1852-1922) was a Croatian nun who is in consideration for beatification by the Vatican, which is facilitated by the identification of her 20th-century remains. Sister Marija was buried in a tomb in Rijeka, Croatia, along with other nuns including her biological sister, Tereza Kozulic (1861-1933). When the remains were exhumed in 2011, they were found in a deteriorated state and commingled with several other sets of remains. Thus, mitochondrial genome sequencing of the long bones was performed to sort the remains by mitochondrial haplotype. Two similar but unique haplotypes belonging to haplogroup H1bu were identified, and samples from these bones were subjected to autosomal short tandem repeat (STR) and single nucleotide polymorphism (SNP) sequencing. Although only partial profiles were obtained, the data were sufficient for kinship analysis with the profile of a paternal niece of Sister Marija (Fides Kozulic). The data indicate that it is 574,195-fold more likely that the two sets of skeletal remains represent 2nd-degree relatives of Fides than sisters who are unrelated to Fides. Although it is impossible to discern which set of remains belongs to Marija and which belongs to Tereza, forensic genomics methods have enabled identification of the sisters.

DNA Testing Reveals the Putative Identity of JB55, a 19th Century Vampire Buried in Griswold, Connecticut.

In 1990 in Griswold, Connecticut, archaeologists excavated a burial found in a "skull and crossbones" orientation. The lid of the 19th century coffin had brass tacks that spelled "JB55", the initials of the person lying there and age at death. JB55 had evidence of chronic pulmonary infection, perhaps tuberculosis. It is possible that JB55 was deemed a vampire due to his disease, and therefore had to be "killed" by mutilating his corpse. In an attempt to reveal the identity of JB55, DNA testing was performed. Ancestry informative single nucleotide polymorphism (SNP) analysis using the Precision ID Ancestry Panel indicated European ancestry. A full Y-chromosomal short tandem repeat (Y-STR) profile was obtained, belonging to haplogroup R1b. When the Y-STR profile was searched in the publicly accessible FamilyTreeDNA R1b Project website, the two closest matches had the surname "Barber". A search of historical records led to a death notice mentioning John Barber, whose son Nathan Barber was buried in Griswold in 1826. The description of Nathan Barber closely fits the burial of "NB13," found near JB55. By applying modern forensic DNA tools to a historical mystery, the identity of JB55 as John Barber, the 19th century Connecticut vampire, has been revealed.

Repair of DNA damage caused by cytosine deamination in mitochondrial DNA of forensic case samples.

DNA sequence damage from cytosine deamination is well documented in degraded samples, such as those from ancient and forensic contexts. This study examined the effect of a DNA repair treatment on mitochondrial DNA (mtDNA) from aged and degraded skeletal samples. DNA extracts from 21 non-probative, degraded skeletal samples (aged 50-70 years) were utilized for the analysis. A portion of each sample extract was subjected to DNA repair using a commercial repair kit, the New England BioLabs' NEBNext FFPE DNA Repair Kit (Ipswich, MA). MtDNA was enriched using PCR and targeted capture in a side-by-side experiment of untreated and repaired DNA. Sequencing was performed using both traditional (Sanger-type; STS) and next-generation sequencing (NGS) methods Although cytosine deamination was evident in the mtDNA sequence data, the observed level of damaged bases varied by sequencing method as well as by enrichment type. The STS PCR amplicon data did not show evidence of cytosine deamination that could be distinguished from background signal in either the untreated or repaired sample set. However, the same PCR amplicons showed 850 C → T/G → A substitutions consistent with cytosine deamination with variant frequencies (VFs) of up to 25% when sequenced using NGS methods The occurrence of base misincorporation due to cytosine deamination was reduced by 98% (to 10) in the NGS amplicon data after repair. The NGS capture data indicated low levels (1-2%) of cytosine deamination in mtDNA fragments that was effectively mitigated by DNA repair. The observed difference in the level of cytosine deamination between the PCR and capture enrichment methods can be attributed to the greater propensity for stochastic effects from the PCR enrichment technique employed (e.g., low template input, increased PCR cycles). Altogether these results indicate that DNA repair may be required when sequencing PCR-amplified DNA from degraded forensic case samples with NGS methods.