Potential applications of nanopore sequencing for forensic analysis.

Advancements in DNA sequencing technologies are occurring at a rapid rate. Various platforms have proven useful in all aspects of health and science research, from molecular diagnostics in cancer research to spore identification in bioterrorism. In the field of forensics, one particular single-molecule sequencing platform shows promise for becoming a viable solution for small to midsize forensic laboratories. Oxford Nanopore Technologies (ONT) has developed a portable, nanopore-based sequencing instrument that has already been utilized for on-site identification of Zika and Ebola viruses, full genome sequencing, evaluation of DNA and RNA base modifications, and enrichment-free mitochondrial DNA analysis. The rapid development of this technology creates possibilities relevant to standard DNA sequencing, direct analysis of forensic samples, including blood, semen, and buccal swabs, mitochondrial DNA analysis, SNP and STR analysis, familial identification, and microbial identification for bioterrorism and geolocation. The small size of the platform, its low cost, and its requirement of only basic laboratory equipment makes this platform well suited for small laboratories wishing to begin developing expertise in sequence-based forensic analyses. Herein, we outline recent developments and applications of nanopore sequencing technologies and their potential application in forensic analysis. We address current and potential techniques in mitochondrial DNA analysis, SNP and STR typing, and microbial identification. Additionally, we discuss recent developments in library preparation and data analysis tool further streamlining the sequencing process that integrate workflows in laboratories or in remote field scenarios.

Deep-Sequencing Technologies and Potential Applications in Forensic DNA Testing.

Development of second- and third-generation DNA sequencing technologies have enabled an increasing number of applications in different areas such as molecular diagnostics, gene therapy, monitoring food and pharmaceutical products, biosecurity, and forensics. These technologies are based on different biochemical principles such as monitoring released pyrophosphate upon incorporation of a base (pyrosequencing), fluorescence detection subsequent to reversible incorporation of a fluorescently labeled terminator base, ligation based approach wherein fluorescence of cleaved nucleotide after ligation is measured, measuring the proton released after incorporation of a base (semiconductor-based sequencing), monitoring incorporation of a nucleotide by measuring the fluorescence of the fluorophore attached to the phosphate chain of the nucleotide, and by detecting the altered charge in a protein nanopore due to released nucleotide by exonuclease cleavage of a DNA strand. Analysis of multiple DNA fragments in parallel increases the depth of coverage while decreasing labor, cost, and time, highlighting some major advantages of deep-sequencing technologies. DNA sequencing has been routinely used in the forensic laboratories for mitochondrial DNA analysis. Fragment analysis, however, is the preferred method for Short Tandem Repeat genotyping due to the cumbersome and costly nature of fi rst-generation DNA sequencing methodologies. Deep-sequencing technologies have brought a new perspective to forensic DNA analysis. Studies include STR analysis to reveal hidden variation in the repeat regions, mtDNA sequencing, Single Nucleotide Polymorphism analysis, mixture resolution, and body fluid identification. Recent publications reveal that attempts are being made to expand the capability.

Hidden Variation in Microsatellite Loci: Utility and Implications for Forensic DNA Analysis.

Short tandem repeat (STR) analysis has been the standard in forensic DNA examinations for almost 15 years. The purpose of this article is to provide some perspective on the biological nature of STR alleles themselves, examine underlying distributions of alleles in the STR loci that are routinely used, and to discuss features of these alleles that are not observable with the currently employed methods. Many of the internationally standardized STR loci contain variations of their interrupted repeat structures, either due to the compound or complex nature of the locus or due to nucleotide variations within the simple repeat motif, which inevitably leads them to become more stratified at the population level. Current STR typing procedures utilizing PCR amplification followed by fragment analysis via capillary or gel electrophoresis does not provide the resolution to discern these polymorphisms. Thus, current designation of alleles is operationally and not biologically defined. Although in the comparison of an evidentiary STR profile to that of a potential contributor, the biological nature of the allele may not be of consequence. When comparisons require assumptions of relatedness between individuals, the biological nature of shared alleles becomes an underlying focus. Herein we will discuss the nature of these additional allelic polymorphisms, what is known of their distribution among the STR loci utilized in forensic testing and within populations, and the advantages this level of allelic discrimination has in forensic and relationship testing.

Single Nucleotide Polymorphisms and Microarray Technology in Forensic Genetics - Development and Application to Mitochondrial DNA.

Variations in the genome, due to base substitutions, insertions, or deletions at single positions, are known as single nucleotide polymorphisms (SNPs). Approximately 85% of human variation is based on such polymorphisms. Therefore, there is an abundance of human SNPs that are available for forensic identity testing purposes. SNP analyses also may be suitable for some forensic identity cases, because they can be detected in smallsized amplicons, allowing for genetic analysis of substantially degraded DNA. While SNP analysis is unlikely to replace short tandem repeat loci typing for routine casework, SNPs may prove useful for certain circumstances, for example, typing mitochondrial DNA (mtDNA). Although sequencing mtDNA enables detection of all SNPs contained within the region of interest, it is currently not a practical approach for simultaneously typing SNPs that reside throughout the entire mtDNA genome. A variety of alternate methods to detect SNPs are available that may facilitate mtDNA analysis. All the methods include amplification, typically by the polymerase chain reaction, of the region containing the SNP of interest. Most assays are based on either hybridization of a probe to amplified product or primer extension chemistry, and multiplexing is possible. Some of these methodologies are: chips, SNaP shot™, Luminex 100™, SNPstream® UHT, and Pyrosequencing™. SNP analysis of mtDNA, both in the noncoding and coding regions, has been demonstrated using a number of these formats.

New host and locality records of coccidia (Apicomplexa: Eimeriidae) from rodents in the southwestern and western United States.

One hundred forty-seven murid and heteromyid rodents were collected from various sites in the southwestern and western United States (Arizona, Colorado, New Mexico, Texas, and Utah) and Baja California Norte, Mexico, and their feces were examined for coccidial parasites. Of these, 53 (36%) were infected with at least 1 coccidian; 45 of 53 (85%) of the infected rodents harbored only 1 species of coccidian. Infected rodents included: 10 of 22 (45%) Neotoma albigula, 3 of 11 (27%) Neotoma floridana, 2 of 14 (14%) Neotoma lepida, 15 of 29 (52%) Neotoma micropus, 5 of 8 (63%) Peromyscus crinitis, 6 of 6 (100%) Peromyscus difficilis, 1 of 2 (50%) Peromyscus eremicus, 9 of 34 (26%) Sigmodon hispidis, and 2 of 3 (67%) Sigmodon ochrognathus; 4 Neotoma cinerea, 3 Neotoma devia, 3 Neotoma mexicana, 1 Peromyscus maniculatus, 1 Onychomys leucogaster, 1 Onychomys torridus, 3 Chaetodipus fallax, and 2 Chaetodipus penicillatus were negative. Although no new species was found, the following coccidians were identified from infected rodents: Eimeria albigulae from N. albigula, N. floridana, and N. micropus, Eimeria antonellii from N. albigula and N. micropus, Eimeria ladronensis from N. albigula, N. floridana, N. lepida, and N. micropus, Eimeria arizonensis and Eimeria lachrymalis from P. crinitis and P. difficilis, Eimeria lachrymalis from P. eremicus, Eimeria tuskeegensis from S. ochrognathus, and Eimeria roperi, Eimeria sigmodontis, Eimeria tuskeegensis, Eimeria webbae, and an unidentified species of Eimeria from S. hispidis. This report documents 12 new host and several distributional records for Eimeria species from murid rodents in Arizona, Texas, and Utah.

A rapid procedure for isolating mitochondrial DNA.

A technique for the rapid isolation of mitochondrial DNA (mtDNA) from animal tissues is described that eliminates the time-consuming separation of nuclear and mtDNAs using cesium chloride gradient ultracentrifugation. The procedure utilizes digestion of the nuclear DNA with DNase, after which lysis of mitochondria and subsequent extraction of proteins results in relatively pure mtDNA. Up to 5 micrograms of mtDNA per gram of liver tissue resulted, a suitable yield for five digests with restriction enzymes and staining with ethidium bromide.