A complete pipeline enables haplotyping and phasing macrohaplotype in long sequencing reads for polyploidy samples and a multi-source DNA mixture.

Macrohaplotype combines multiple types of phased DNA variants, increasing forensic discrimination power. High-quality long-sequencing reads, for example, PacBio HiFi reads, provide data to detect macrohaplotypes in multiploidy and DNA mixtures. However, the bioinformatics tools for detecting macrohaplotypes are lacking. In this study, we developed a bioinformatics software, MacroHapCaller, in which targeted loci (i.e., short TRs [STRs], single nucleotide polymorphisms, and insertion and deletions) are genotyped and combined with novel algorithms to call macrohaplotypes from long reads. MacroHapCaller uses physical phasing (i.e., read-backed phasing) to identify macrohaplotypes, and thus it can detect multi-allelic macrohaplotypes for a given sample. MacroHapCaller was validated with data generated from our designed targeted PacBio HiFi sequencing pipeline, which sequenced ∼8-kb amplicon regions harboring 20 core forensic STR loci in human benchmark samples HG002 and HG003. MacroHapCaller also was validated in whole-genome long-read sequencing data. Robust and accurate genotyping and phased macrohaplotypes were obtained with MacroHapCaller compared with the known ground truth. MacroHapCaller achieved a higher or consistent genotyping accuracy and faster speed than existing tools HipSTR and DeepVar. MacroHapCaller enables efficient macrohaplotype analysis from high-throughput sequencing data and supports applications using discriminating macrohaplotypes.

Unraveling crime scenes strand by strand: the forensic odyssey of Bruce Budowle.

Bruce Budowle speaks to Ashling Cannon, Journal Development Editor for BioTechniques, about advancements & challenges in forensic science. Budowle completed his doctorate in genetics at Virginia Tech (VA, USA) formally known as Virginia Polytechnic Institute and State University. He then went on to complete a postdoctoral fellowship at the University of Alabama at Birmingham (AL, USA) to study genetic risk factors for acute lymphocytic leukemia, diabetes and melanoma. Budowle was early in his career and hadn't spent much time in forensics at this stage, but in 1982 an advert caught his eye for a job with the FBI to develop genetic marker systems to identify people who have left biological evidence at crime scenes. Budowle spent 26 years with the FBI and helped develop a plethora of genetic analysis methods. In 1985, it became a reality that DNA could be a signature for identifying people, and there were huge developments in DNA forensic analysis. In 2009, Budowle moved into academia and went to the University of North Texas Health Science Center (TX, USA), eventually becoming the Director of the Center for Human Identification, where he oversaw missing person and traditional crime cases, taught students and carried out fundamental and applied research. Budowle feels incredibly lucky to have had the resources, opportunities and academic infrastructure to learn and develop his knowledge. Budowle recently retired from academia and now spends his time building capacity for DNA forensics applications in Africa through the Department of Justice, with a well-established program known as the International Criminal Investigative Training Assistance Program (ICITAP) as well as with the non-government organization (NGO) DNAforAfrica.

Techniques for estimating genetically variable peptides and semi-continuous likelihoods from massively parallel sequencing data.

Forensic genetic investigations typically rely on analysis of DNA for attribution purposes. There are times, however, when the amount and/or the quality of the DNA is limited, and thus little or no information can be obtained regarding the source of the sample. An alternative biochemical target that also contains genetic signatures is protein. One class of genetic signatures is protein polymorphisms that are a direct consequence of simple/single/short nucleotide polymorphisms (SNPs) in DNA. However, to interpret protein polymorphisms in a forensic context, certain complexities must be understood and addressed. These complexities include: 1) SNPs can generate 0, 1, or arbitrarily many polymorphisms in a polypeptide; and 2) as an object of expression that is modulated by alleles, genes and interactions with the environment, proteins may be present or absent in a given sample. To address these issues, a novel approach was taken to generate the expected protein alleles in a reference sample based on whole genome (or exome) sequence data and assess the significance of the evidence using a haplotype-based semi-continuous likelihood algorithm that leverages whole proteome data. Converting the genomic information into the proteomic information allows for the zero-to-many relationship between SNPs and GVPs to be abstracted away. When viewed as a haplotype, many GVPs that correspond to the same SNP is equivalent to many SNPs in perfect linkage disequilibrium (LD). As long as the likelihood formulation correctly accounts for LD, the correspondence between the SNP and the proteome can be safely neglected. Tests were performed on simulated samples, including single-source and two-person mixtures, and the power of using a classical semi-continuous likelihood versus one that has been adapted to neglect drop-out was compared. Additionally, summary statistics and a rudimentary set of decision guidelines were introduced to help identify mixtures from protein data.

An algorithm for random match probability calculation from peptide sequences.

For the past three decades, forensic genetic investigations have focused on elucidating DNA signatures. While DNA has a number of desirable properties (e.g., presence in most biological materials, an amenable chemistry for analysis and well-developed statistics), DNA also has limitations. DNA may be in low quantity in some tissues, such as hair, and in some tissues it may degrade more readily than its protein counterparts. Recent research efforts have shown the feasibility of performing protein-based human identification in cases in which recovery of DNA is challenged; however, the methods involved in assessing the rarity of a given protein profile have not been addressed adequately. In this paper an algorithm is proposed that describes the computation of a random match probability (RMP) resulting from a genetically variable peptide signature. The approach described herein explicitly models proteomic error and genetic linkage, makes no assumptions as to allelic drop-out, and maps the observed proteomic alleles to their expected protein products from DNA which, in turn, permits standard corrections for population structure and finite database sizes. To assess the feasibility of this approach, RMPs were estimated from peptide profiles of skin samples from 25 individuals of European ancestry. 126 common peptide alleles were used in this approach, yielding a mean RMP of approximately 10-2.

A genome-wide association study of tramadol metabolism from post-mortem samples.

Phase I tramadol metabolism requires cytochrome p450 family 2, subfamily D, polypeptide 6 (CYP2D6) to form O-desmethyltramadol (M1). CYP2D6 genetic variants may infer metabolizer phenotype; however, drug ADME (absorption, distribution, metabolism, and excretion) and response depend on protein pathway(s), not CYP2D6 alone. There is a paucity of data regarding the contribution of trans-acting proteins to idiosyncratic phenotypes following drug exposure. A genome-wide association study identified five markers (rs79983226/kgp11274252, rs9384825, rs62435418/kgp10370907, rs72732317/kgp3743668, and rs184199168/exm1592932) associated with the conversion of tramadol to M1 (M1:T). These SNPs reside within five genes previously implicated with adverse reactions. Analysis of accompanying toxicological meta-data revealed a significant positive linear relationship between M1:T and degree of sample polypharmacy. Taken together, these data identify candidate loci for potential clinical inferences of phenotype following exposure to tramadol and highlight sample polypharmacy as a possible diagnostic covariate in post-mortem genetic studies.

A pathway-driven predictive model of tramadol pharmacogenetics.

Predicting metabolizer phenotype (MP) is typically performed using data from a single gene. Cytochrome p450 family 2 subfamily D polypeptide 6 (CYP2D6) is considered the primary gene for predicting MP in reference to approximately 30% of marketed drugs and endogenous toxins. CYP2D6 predictions have proven clinically effective but also have well-documented inaccuracies due to relatively high genotype-phenotype discordance in certain populations. Herein, a pathway-driven predictive model employs genetic data from uridine diphosphate glucuronosyltransferase, family 1, polypeptide B7 (UGT2B7), adenosine triphosphate (ATP)-binding cassette, subfamily B, number 1 (ABCB1), opioid receptor mu 1 (OPRM1), and catechol-O-methyltransferase (COMT) to predict the tramadol to primary metabolite ratio (T:M1) and the resulting toxicologically inferred MP (t-MP). These data were then combined with CYP2D6 data to evaluate performance of a fully combinatorial model relative to CYP2D6 alone. These data identify UGT2B7 as a potentially significant explanatory marker for T:M1 variability in a population of tramadol-exposed individuals of Finnish ancestry. Supervised machine learning and feature selection were used to demonstrate that a set of 16 loci from 5 genes can predict t-MP with over 90% accuracy, depending on t-MP category and algorithm, which was significantly greater than predictions made by CYP2D6 alone.

Supervised Classification of CYP2D6 Genotype and Metabolizer Phenotype With Postmortem Tramadol-Exposed Finns.

Cytochrome p450 family 2, subfamily D, polypeptide 6 (CYP2D6) may be used to infer the metabolizer phenotype (MP) of an individual as poor, intermediate, extensive/normal, or ultrarapid. Metabolizer phenotypes may suggest idiosyncratic drug responses as contributing factors to cause and/or manner of death in postmortem investigations. Application of CYP2D6 has used long-range amplification of the locus and restriction enzyme digestion to detect single-nucleotide variants (SNVs) associated with MPs. This process can be cumbersome and requires knowledge of genotype phase. Phase may be achieved using long-read DNA sequencing and/or computational methods; however, both can be error prone, which may make it difficult or impractical for implementation into medicolegal practice. CYP2D6 was interrogated in postmortem autopsied Finns using supervised machine learning and feature selection to identify SNVs indicative of MP and/or rate of tramadol O-demethylation (T:M1). A subset of 18 CYP2D6 SNVs could predict MP/T:M1 with up to 96.3% accuracy given phased data. These data indicate that phase contributes to classification accuracy when using CYP2D6 data. Of these 18 SNVs, 3 are novel loci putatively associated with T:M1. These findings may enable design of small multiplexes for easy forensic application of MP prediction when cause and/or manner of death is unknown.

Predicted activity of UGT2B7, ABCB1, OPRM1, and COMT using full-gene haplotypes and their association with the CYP2D6-inferred metabolizer phenotype.

The pharmacogene, CYP2D6, is commonly used to infer metabolizer phenotype of many marketed drugs and endogenous toxins in ante- and post-mortem patients but only represents the efficiency of phase 1 metabolism. Downstream metabolic enzymes encoded by UGT2B7, ABCB1, OPRM1, and COMT also have been implicated in variable individual response to drugs due to their activity at different stages of the tramadol ADME (absorption, distribution, metabolism, and excretion) process. While commonly studied as single genes using targeted genotyping approaches, a more comprehensive tramadol metabolism profile has not been evaluated. 1000 Genomes Project data for UGT2B7, ABCB1, OPRM1, and COMT were used to characterize full-gene haplotypes and their effect on protein function using in-house excel-based workbooks, PopART, and TreeView. Population genetic summary statistics and intergenic analyses associated these haplotypes with full-gene CYP2D6-inferred metabolizer phenotype. The findings suggest that UGT2B7, ABCB1, OPRM1, and COMT may contribute to predicted metabolizer phenotype as opposed to relying solely on CYP2D6.

Internal validation of the RapidHIT® ID system.

Traditionally, forensic DNA analysis has required highly skilled forensic geneticists in a dedicated laboratory to generate short tandem repeat (STR) profiles. STR profiles are routinely used either to associate or exclude potential donors of forensic biological evidence. The typing of forensic reference samples has become more demanding, especially with the requirement in some jurisdictions to DNA profile arrestees. The Rapid DNA (RDNA) platform, the RapidHIT® ID (IntegenX®, Pleasanton, CA), is a fully automated system capable of processing reference samples in approximately 90min with minimal human intervention. Thus, the RapidHIT ID instrument can be deployed to non-laboratory environments (e.g., booking stations) and run by trained atypical personnel such as law enforcement. In order to implement the RapidHIT ID platform, validation studies are needed to define the performance and limitations of the system. Internal validation studies were undertaken with four early-production RapidHIT ID units. Reliable and concordant STR profiles were obtained from reference buccal swabs. Throughout the study, no contamination was observed. The overall first-pass success rate with an "expert-like system" was 72%, which is comparable to another current RDNA platform commercially available. The system's second-pass success rate (involving manual interpretation on first-pass inconclusive results) increased to 90%. Inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect typing by the instrument system; however, substrate (i.e., swab type) did impact typing success. Additionally, one desirable feature not available with other Rapid systems is that in the event of a system failed run, a swab can be recovered and subsequently re-analyzed in a new sample cartridge. Therefore, rarely should additional sampling or swab consumption be necessary. The RapidHIT ID system is a robust and reliable tool capable of generating complete STR profiles within the forensic DNA typing laboratory or with proper training in decentralized environments by non-laboratory personnel.

General Aviation Pilots Over 70 Years Old.

BACKGROUND: Currently it is not unusual for general aviation pilots in the United States to continue to fly beyond the age of 70, even into their 80s and 90s. Pilots have regular examinations according to protocols which do not specify special or additional requirements for pilots over 70 yr of age. Additionally, the third class medical reforms passed by the U.S. Senate on 15 July 2016 could potentially result in even less stringent medical certification requirements for general aviation pilots. METHODS: Accident rates, medical parameters, autopsy findings, and toxicological findings from the U.S. National Transportation Safety Board (NTSB) general aviation (GA) accident database were analyzed to assess potential risk factors with accident outcomes. RESULTS: During 2003-2012, there were 114 (113 men, 1 woman) general aviation fatal accidents involving pilots ages 70 to 92 yr. A combination of 3 or more drugs were found in 13 (13%) of deceased pilots. The most frequent drugs were first generation antihistamines and antidepressants represented the next highest proportion of possible performance-affecting medications. CONCLUSION: This study indicates that there are critical medical factors that may contribute to fatal accidents among elderly pilots. Polypharmacy use should be taken into consideration, especially during periodic health examinations and fatal aviation investigations involving elderly pilots.Vuorio A, Asmayawati S, Budowle B, Griffiths R, Strandberg T, Kuoppala J, Sajantila A. General aviation pilots over 70 years old. Aerosp Med Hum Perform. 2017; 88(2):142-145.

Paternity calculations in a di-spermy case.

In a criminal paternity case, which involved analysis of the product of conception, a rare circumstance was observed. The product of conception was triploidy, apparently due to an egg fertilized by two sperm. Since there is little guidance on how to calculate the probability of the DNA evidence given some basic hypotheses, the formulae were derived and are presented herein. These approaches could provide guidance for similar situations if they arise.

Enhancement of Pathologist's Routine Practice: Reuse of DNA Extracted from Immunostained Formalin-fixed Paraffin-embedded (FFPE) Slides in Downstream Molecular Analysis of Cancer.

BACKGROUND/AIM: To date, the conventional formalin-fixed, paraffin-embedded (FFPE) technique is the gold-standard for preserving histomorphology. Once FFPE tissues are stained, slides are routinely archived along with their blocks at biobanks/hospitals. However, the reuse of fixed and stained biospecimens as DNA source is not a common routine practice worldwide and, thus, indicates the need of studies to investigate the feasibility of extracting DNA from already immunohistochemistry (IHC) FFPE-stained slides and its possible reuse in subsequent downstream molecular analyses. MATERIALS AND METHODS: FFPE IHC slides from colorectal cancer (CRC) patients were prepared and stored in the CEGMR Biobank. The workflow consists of digitalization of IHC stained slide's image, removing the slide cover-slip, crude dissection and DNA extraction. Following DNA quality assessment, mutation analysis of CTNNB1 and methylation profile of CDH1 were performed. RESULTS: High-quality DNA was obtained allowing 60% concordance between CDH1 methylation and membranous E-cadherin expression pattern. Clean CTNNB1 DNA chromatograms with evenly-spaced peaks were observed. CONCLUSION: This study is a proof of concept to recycle and reuse DNA from IHC stained slides with suitable concentration and integrity for further downstream molecular applications. These findings will enhance the pathologists' knowledge, attitudes and practices (KAP) towards the use of these biospecimens and support the implementation of this approach in clinical pathology practice. Therefore, the scientific community will benefit from the largest comprehensive database of human fully annotated FFPE biospecimens already available at their disposal in order to demystify the complexity and the heterogeneity of many challenging diseases and foster the transition towards precision medicine.

Utility of amplification enhancers in low copy number DNA analysis.

One parameter that impacts the robustness and reliability of forensic DNA analyses is the amount of template DNA used in the polymerase chain reaction (PCR). With short tandem repeat (STR) typing, low copy number (LCN) DNA samples can present exaggerated stochastic effects during the PCR that result in heterozygote peak height imbalance, allele drop out, and increased stutter. Despite these effects, there has been little progress toward decreasing the formation of stutter products and heterozygote peak imbalance effects during PCR. In an attempt to develop a more robust system that is less refractory to stochastic effects, the PCR additives, betaine, DMSO, PEG, and PCRboost®, were investigated on low-quantity DNA samples. The effects of the additives were assessed by evaluating STR typing results. Of the four additives, the only positive effects were observed with betaine treatment. Betaine, at a final concentration of 1.25 mol/L, was found to improve the robustness of the amplification, specifically by decreasing stutter in a dual locus system. In contrast, the addition of 1.25 mol/L betaine to commercial STR amplification kits did not affect stutter ratios. However, the addition of betaine did lead to increased yield of PCR products in all commercial kits tested. The results support that betaine can improve amplification efficiency of LCN DNA samples.

An evaluation of the transfer of saliva-derived DNA.

Studies of DNA transfer have focused largely on the transfer of sloughed off epithelial cells from individuals' hands. This research examines primary, secondary, and tertiary transfer events involving DNA originating from saliva, a commonly encountered body fluid. More routine human behaviors were simulated to evaluate transfer, and the effects of drying time, moisture, and surface composition were investigated. The results agree with previous findings which indicate that the presence of moisture, as well as a smooth nonporous surface as the primary substrate, increases the efficiency of transfer. Previous transfer studies have found that the last individual to come into contact with an item is usually the major contributor to the resulting DNA mixture, unless conditions are simulated in which a "good shedder" serves as the primary depositor and a poor shedder serves as the secondary depositor. The results of this study indicate that when saliva is the source of the transferred DNA, the primary depositor is often the major contributor. These findings suggest that shedder status is less relevant with regard to touch DNA samples in a forensic setting and emphasize the need for caution when analyzing such samples.

Extraction platform evaluations: a comparison of AutoMate Express™, EZ1® Advanced XL, and Maxwell® 16 Bench-top DNA extraction systems.

The DNA extraction performance of three low-throughput extraction systems was evaluated. The instruments and respective chemistries all use a similar extraction methodology that involves binding DNA to a coated magnetic resin in the presence of chaotropic salt, washing of the resin to remove undesirable compounds, and elution of DNA from the particles in a low-salt solution. The AutoMate Express™ (Life Technologies Corporation, Carlsbad, CA), EZ1® Advanced XL (Qiagen Inc., Valencia, CA), and Maxwell® 16 (Promega Corporation, Madison, WI) were compared using a variety of samples including: blood on swabs, blood on denim, blood on cotton, blood mixed with inhibitors (a mixture of indigo, hematin, humic acid, and urban dust) on cotton, blood on FTA® paper, saliva residue on cigarette butt paper, epithelial cells on cotton swabs, neat semen on cotton, hair roots, bones, and teeth. Each instrument had a recommended pre-processing protocol for each sample type, and these protocols were followed strictly to reduce user bias. All extractions were performed in triplicate for each sample type. The three instruments were compared on the basis of quantity of DNA recovered (as determined by real-time PCR), relative level of inhibitors present in the extract (shown as shifts in the C(T) value for the internal PCR control in the real-time PCR assay), STR peak heights, use of consumables not included in the extraction kits, ease of use, and application flexibility. All three systems performed well; however extraction efficiency varied by sample type and with the preprocessing protocol applied to the various samples.

HIV legal precedent useful for microbial forensics.

The field of microbial forensics was formalized because of the need for attribution in events where a bioweapon has been used. Microbial forensics has its origins in traditional forensics, microbiology, and epidemiology. Microbial forensics can be defined as a scientific discipline dedicated to analyzing evidence for attribution purposes from a bioterrorism act, biocrime, hoax, or inadvertent microorganism/toxin release. This is a very challenging task, since there are myriad microorganisms that can pose a threat, and analytical methods need to be used reliably. The Scientific Working Group on Microbial Genetics and Forensics (SWGMGF) has addressed some quality assurance and control issues, and particularly validation criteria (focusing on preliminary validation) due to the dynamic nature of evolving investigations. Unique identification of a microorganism may never be possible. Yet, qualitative and/or quantitative assessments of the evidence can be made. One approach to provide direction on gaps in the microbial forensics effort is to perform an end-to-end retrospective analysis of past cases. As an example, the case of a gastroenterologist who was accused of second degree attempted murder of his paramour using HIV as the weapon was reviewed. The scientific evaluation involves epidemiology, molecular biology, phylogenetics, and legal deliberations.