Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

Methods used in microbial forensics and epidemiological investigations for stronger health systems.

This review discusses microbial forensics as an emerging science that finds application in protecting human health. It is important to distinguish naturally acquired infections from those caused by the intentional release of microorganisms to the environment. This information is crucial in formulating procedures against the spread of infectious diseases and prosecuting persons who may be involved in acts of biocrime, bioterrorism, or biowarfare. A comparison between epidemiological investigations and microbial forensic investigations is provided. In addition, a discussion on how microbial forensics strengthens health systems is included in this review. Microbial forensic investigations and epidemiologic examinations employ similar concepts and involve identifying and characterising the microbe of interest. Both fields require formulating an appropriate case definition, determining a pathogen's mode of transmission, and identifying the source(s) of infection. However, the two subdisciplines differ in their objectives. An epidemiological investigation aims to identify the pathogen's source to prevent the spread of the disease. Microbial forensics focuses on source-tracking to facilitate the prosecution of persons responsible for the spread of a pathogen. Both fields use molecular techniques in analysing and comparing DNA, gene products, and biomolecules to identify and characterise the microorganisms of interest. We included case studies to show methods used in microbial forensic investigations, a brief discussion of the public significance of microbial forensic systems, and a roadmap for establishing a system at a national level. This system is expected to strengthen a country's capacity to respond to public health emergencies. Several factors must be considered in establishing national microbial forensic systems. First is the inherent ubiquity, diversity, and adaptability of microorganisms that warrants the use of robust and accurate molecular typing systems. Second, the availability of facilities and scientists who have been trained in epidemiology, molecular biology, bioinformatics, and data analytics. Human resources and infrastructure are critical requirements because formulating strategies and allocating resources in times of infectious disease outbreaks must be data-driven. Establishing and maintaining a national microbial forensic system to strengthen capacities in conducting forensic and epidemiological investigations should be prioritised by all countries, accompanied by a national policy that sets the legislative framework and provides for the system's financial requirements.

Ethical challenges in genetic research among Philippine Indigenous Peoples: Insights from fieldwork in Zamboanga and the Sulu Archipelago.

The Philippines, with the recent discovery of an archaic hominin in Luzon and an extensive ethnolinguistic diversity of more than 100 Indigenous peoples, is crucial to understanding human evolution and population history in Island Southeast Asia. Advances in DNA sequencing technologies enable the rapid generation of genomic data to robustly address questions about origins, relatedness, and population movements. With the increased genetic sampling in the country, especially by international scientists, it is vital to revisit ethical rules and guidelines relevant to conducting research among Indigenous peoples. Our team led fieldwork expeditions between 2019 and February 2020 in Zamboanga and the Sulu Archipelago, a chain of islands connecting the Mindanao and Borneo landmasses. The trips concluded with a collection of 2,149 DNA samples from 104 field sites. We present our fieldwork experience among the mostly sea-oriented Sama-Bajaw and Tausug-speaking communities and propose recommendations to address the ethical challenges of conducting such research. This work contributes toward building an enabling research environment in the Philippines that respects the rights and autonomy of Indigenous peoples, who are the rightful owners of their DNA and all genetic information contained therein.

Philippine mitochondrial DNA diversity: a populated viaduct between Taiwan and Indonesia?

Relatively little is known about the genetic diversity of the Philippine population, and this is an important gap in our understanding of Southeast Asian and Oceanic prehistory. Here we describe mitochondrial DNA (mtDNA) variation in 423 Philippine samples and analyze them in the context of the genetic diversity of other Southeast Asian populations. The majority of Philippine mtDNA types are shared with Taiwanese aboriginal groups and belong to haplogroups of postglacial and pre-Neolithic origin that have previously been identified in East Asian and Island Southeast Asian populations. Analysis of hypervariable segment I sequence variation within individual mtDNA haplogroups indicates a general decrease in the diversity of the most frequent types (B4a1a, E1a1a, and M7c3c) from the Taiwanese aborigines to the Philippines and Sulawesi, although calculated standard error measures overlap for these populations. This finding, together with the geographical distribution of ancestral and derived haplotypes of the B4a1a subclade including the Polynesian Motif, is consistent with southward dispersal of these lineages "Out of Taiwan" via the Philippines to Near Oceania and Polynesia. In addition to the mtDNA components shared with Taiwanese aborigines, complete sequence analyses revealed a minority of lineages in the Philippines that share their origins--possibly dating back to the Paleolithic--with haplogroups from Indonesia and New Guinea. Other rare lineages in the Philippines have no closely related types yet identified elsewhere.

Y-STR DNA analysis of 154 female child sexual assault cases in the Philippines.

The laboratory evaluated 154 sexual assault cases from four Child Protection Units in the Philippines involving female child victims aged from 2 years to 18 years old. All child victims sought medical attention within 72 h after sexual contact. In 130 cases, the child victim knew the alleged offender and identified them during the interview with the social worker. Penile ejaculation was reported by 68 child victims with varying reports of washing after contact. Overall, 84 child victims admitted having wiped their genitalia prior to the collection of biological samples for DNA testing. Laboratory personnel examined vaginal smears in only 109 cases using a light microscope and reported 23 samples to be positive for sperm cells. Using the PowerPlex® short tandem repeat of the Y chromosome (Y-STR) DNA multiplex system, male DNA was detected in vaginal swab samples from 63 child victims. In 39 cases, positive amplification at 11 Y-STR DNA markers consistent with a single male DNA profile was observed. Twenty-eight of these full single Y-STR DNA profiles were found to be unique when searched in worldwide Y-STR DNA population databases (~40,000 haplotypes), eight haplotypes matching Filipinos and/or Asian haplotypes and one Y-STR DNA profile only matching European, Caucasian, and Latin American haplotypes. Y-STR DNA profiles generated will be compared with reference DNA profiles of alleged offenders once reference samples are submitted to the laboratory.

Death in the time of Covid-19: Efforts to restore the death penalty in the Philippines.

The Philippine Congress recently passed a bill amending the Dangerous Drugs Act of 2002 and reimposing the penalty of life imprisonment to death for specific-drug related offenses. House Bill No. 7814 also allows the presumption of guilt in certain drug-related crimes unless otherwise proven, thereby overturning the long-standing constitutional presumption of innocence. The bill has been sent to the Senate for its concurrence and could only be several steps away before being signed into law by President Rodrigo R. Duterte. This paper discusses the ramifications of the new bill and the questioned timeliness of its passage when the country continues to have a large and overcrowded prison population and a significant number of deaths due to SARS-CoV-2 in Southeast Asia. The government's lapses in following the 2021 national vaccination plan became apparent in the 31 March 2021 assessment made by the congressional health panel on the government's response to the pandemic. From the authors' perspective, the urgency of using the country's limited resources to help medical frontliners and local government units prevent further infections and save lives should have outweighed the efforts exerted to pass a law that legalized the death penalty for the third time in the Philippines.

Improved autosomal STR typing of degraded femur samples extracted using a custom demineralization buffer and DNA IQ™.

Bone samples are utilized as a source of DNA in disaster victim identification and human remains investigations. However, DNA recovery from bones is time consuming and prone to contamination. A logical approach for postmortem identification is to validate efficient DNA extraction methods requiring less bone material using improved molecular kits with less hands-on time and workflows that facilitate faster turn-around time for processing. In this study, we evaluated DNA yield and amplification efficiency of DNA extracts using a new custom bone demineralization buffer (DMB; Promega) and extracted via manual and automated DNA IQ™ workflows. Including the demineralization step, the bone protocol can be completed in ∼4 h and even less with minimal sample handling when automated. Overall, a rapid and simple DNA extraction with improved allele recovery was validated using degraded bone samples exposed to tropical environments and post-disaster recovery as well as adverse conditions of embalming prior to internment.

An integrated system for forensic DNA testing of sexual assault cases in the Philippines.

In the Philippines, more than 7000 cases of sexual assault are reported annually. DNA technology is a powerful tool in identifying assailants. However, it is not routinely used in sexual assault investigations due to insufficient government support to cover the high cost of DNA testing and the absence of a national system for sample collection, handling, storage, and DNA testing of biological evidence. In itself, the nature of sexual assault samples containing DNA mixtures presents challenges to laboratory methods and interpretation of results. The sample recovered from the victim may only contain trace amounts of the assailant's DNA, may have degraded due to prolonged storage in ambient conditions which is warm and humid in the tropics, or contaminated with inhibitors, such as in anal swabs. Hence, a closer evaluation of the processes of evidence collection and DNA testing is needed to increase the likelihood of success in generating conclusive results. In this paper, we propose an integrated system for DNA testing of biological samples collected from sexual assault victims considering the limitations of resources and the prevailing warm climate. Recommendations in this work should provide basis for formulating national guidelines for DNA analysis in aid of criminal investigations. The proposed scheme can be adopted by forensic DNA laboratories in the Philippines and in other countries facing similar challenges.

Forensic DNA testing during the SARS-CoV-2 pandemic.

The aggressive nature of the new SARS-2 corona virus now referred to as SARS-CoV-2 ; the seriousness and length of the period of infection; the fast and far-reaching transmissibility via liquid droplets that become air-borne when someone coughs, sneezes or speaks with increasing evidence to support actual airborne transmission; the presence of viral particles especially in body fluids and tissues, of viral positive individuals; and the persistence of the virus on different types of surfaces pose serious concerns for forensic practitioners, including forensic DNA analysts. Many forensic laboratories and law enforcement agencies need to address the inevitable changes that must be made in forensic DNA testing. In this article, we explore the effects of the COVID-19 pandemic on the collection, handling, storage and transport of biological samples for downstream DNA testing. This paper aims to open discussions on the urgency of balancing the need to conduct investigations in order to maintain public order with the requirements of effective biosafety protocols specifically formulated to protect human resources within the forensic science community.

The war on drugs, forensic science and the death penalty in the Philippines.

The effectiveness of the death penalty to deter heinous crimes remains a contentious issue even though it has been abolished in many countries. Three years into President Rodrigo Duterte's administration, the push to re-impose the death penalty is being taken seriously. There is urgency in providing options to the drug problem other than killing drug suspects in the streets or sentencing them to death. The drug problem is a complex issue and exposes the human vulnerability of its users for criminal exploitation. We propose here that addressing these vulnerabilities in a balanced and comprehensive manner through health-focused, rights-based criminal justice responses, conducting forensic science-based drug investigations and determining the social causes of drug abuse is an alternative solution that demands cooperation across different sectors of society as well as underscores the fundamental value of human life.

Data on likelihood ratios of two-person DNA mixtures interpreted using semi- and fully continuous systems.

In the paper, "Probabilistic approaches to interpreting two-person DNA mixtures from post-coital specimens" [1], we analysed 102 two-person DNA samples from simulated mixtures and male-female and male-male post-coital specimens. We report here data on profile characteristics of these samples and likelihood ratios (LRs) generated using semi- and fully continuous systems. Both log10 LRs from true and non-contributor tests are presented. These data may supplement studies comparing performance of different probabilistic systems for DNA evidence interpretation.

Probabilistic approaches to interpreting two-person DNA mixtures from post-coital specimens.

Biological samples submitted for sexual assault investigation typically involve mixtures of DNA from the victim and the assailant/s. Providing a statistical weight to such evidence may be mathematically complex and may be affected by subjective judgment of a human analyst. Software tools have been developed to address these issues. To contribute towards improving the system for routine DNA testing of sexual assault cases, we evaluated two likelihood ratio (LR) approaches: a semi-continuous model using LRmix Studio and a fully continuous approach employed in STRmix™ for interpreting two-person DNA mixtures. LRs conditioned on the presence of the receptive partner's DNA were calculated for a total of 102 two-person DNA samples from simulated mixtures and various post-coital samples. Our results highlight the importance of maximising information provided into the LR calculation to generate strong support for the true hypothesis. This can be achieved by recovering sufficient DNA from a sample to minimise risk of drop-out and increase peak intensities and by implementing a statistical model that utilises as much of the electropherogram information as possible. LRmix is open-source and can handle profiles with allelic drop-out and drop-ins, however stuttering is not modelled and requires manual removal by a DNA analyst especially for mixtures with low template components. STRmix™ makes effective use of all available information by incorporating into its biological model complicating aspects of a DNA profile such as degradation, allele drop-out and drop-in, stutters, and peak height variability.

Population data of 10 Y-chromosomal STR Loci in Cebu province, Central Visayas (Philippines).

POPULATION: 103 unrelated male individuals from Cebu province, Central Visayas region of the Philippines.

Filipino DNA variation at 12 X-chromosome short tandem repeat markers.

Demands for solving complex kinship scenarios where only distant relatives are available for testing have risen in the past years. In these instances, other genetic markers such as X-chromosome short tandem repeat (X-STR) markers are employed to supplement autosomal and Y-chromosomal STR DNA typing. However, prior to use, the degree of STR polymorphism in the population requires evaluation through generation of an allele or haplotype frequency population database. This population database is also used for statistical evaluation of DNA typing results. Here, we report X-STR data from 143 unrelated Filipino male individuals who were genotyped via conventional polymerase chain reaction-capillary electrophoresis (PCR-CE) using the 12 X-STR loci included in the Investigator® Argus X-12 kit (Qiagen) and via massively parallel sequencing (MPS) of seven X-STR loci included in the ForenSeq™ DNA Signature Prep kit of the MiSeq® FGx™ Forensic Genomics System (Illumina). Allele calls between PCR-CE and MPS systems were consistent (100% concordance) across seven overlapping X-STRs. Allele and haplotype frequencies and other parameters of forensic interest were calculated based on length (PCR-CE, 12 X-STRs) and sequence (MPS, seven X-STRs) variations observed in the population. Results of our study indicate that the 12 X-STRs in the PCR-CE system are highly informative for the Filipino population. MPS of seven X-STR loci identified 73 X-STR alleles compared with 55 X-STR alleles that were identified solely by length via PCR-CE. Of the 73 sequence-based alleles observed, six alleles have not been reported in the literature. The population data presented here may serve as a reference Philippine frequency database of X-STRs for forensic casework applications.

Allele frequencies for two pentanucleotide STR loci Penta D and Penta E in a Philippine population.

Allele frequencies for the pentanucleotide STR markers Penta D and Penta E were analyzed in 100 unrelated Filipinos from different regions of the Philippine archipelago. These markers were found to be highly polymorphic in the studied population.

Comparison of Two Massively Parallel Sequencing Platforms using 83 Single Nucleotide Polymorphisms for Human Identification.

The potential of Massively Parallel Sequencing (MPS) technology to vastly expand the capabilities of human identification led to the emergence of different MPS platforms that use forensically relevant genetic markers. Two of the MPS platforms that are currently available are the MiSeq® FGx™ Forensic Genomics System (Illumina) and the HID-Ion Personal Genome Machine (PGM)™ (Thermo Fisher Scientific). These are coupled with the ForenSeq™ DNA Signature Prep kit (Illumina) and the HID-Ion AmpliSeq™ Identity Panel (Thermo Fisher Scientific), respectively. In this study, we compared the genotyping performance of the two MPS systems based on 83 SNP markers that are present in both MPS marker panels. Results show that MiSeq® FGx™ has greater sample-to-sample variation than the HID-Ion PGM™ in terms of read counts for all the 83 SNP markers. Allele coverage ratio (ACR) values show generally balanced heterozygous reads for both platforms. Two and four SNP markers from the MiSeq® FGx™ and HID-Ion PGM™, respectively, have average ACR values lower than the recommended value of 0.67. Comparison of genotype calls showed 99.7% concordance between the two platforms.

Development of a pentaplex X-chromosomal short tandem repeat typing system and population genetic studies.

Quadruplex and pentaplex systems for polymerase chain reaction amplification of X-chromosomal short tandem repeats DXS101, HPRTB, DXS8377, DXS981 (STRX1) and DXS6789 were developed for automated profiling of liquid and membrane-bound DNA samples. Chinese, Japanese and Thai populations were typed using a quadruplex system, while German and Philippine populations were analyzed using a five-locus system. Out of 88 meioses studied in Philippine family samples at each locus, a possible one repeat deletion (allele 51 to 50) at DXS8377 was observed in a father-daughter pair. Exact tests performed on genotype data from females in the Philippine, German and Thai populations indicated that these groups conform to Hardy-Weinberg equilibrium. Exact tests for population differentiation indicate significant variations in allele distributions, particularly at loci DXS101, DXS981 and DXS6789. Considered individually, DXS8377 was the most polymorphic and HPRTB the least polymorphic locus in these five populations. When the forensic efficiency of the quadruplex system was calculated, the combined power of discrimination among males (PD(M)) was no lower than 0.998, while among females the combined PD(F) was at least 0.9999 in all populations. The combined power of paternity exclusion was a minimum of 0.998 in trio cases and 0.98 in motherless cases. The addition of locus DXS6789 to the German and Philippine population databases using a pentaplex increased the forensic efficiency of the analysis system.