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.

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.

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.

Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification.

The capability of DNA laboratories to perform genotyping procedures from post-mortem remains, including those that had undergone putrefaction, continues to be a challenge in the Philippines, a country characterized by very humid and warm conditions all year round. These environmental conditions accelerate the decomposition of human remains that were recovered after a disaster and those that were left abandoned after a crime. When considerable tissue decomposition of human remains has taken place, there is no other option but to extract DNA from bone and/or teeth samples. Routinely, femur shafts are obtained from recovered bodies for human identification because the calcium matrix protects the DNA contained in the osteocytes. In the Philippines, there is difficulty in collecting femur samples after natural disasters or even human-made disasters, because these events are usually characterized by a large number of fatalities. Identification of casualties is further delayed by limitation in human and material resources. Hence, it is imperative to test other types of biological samples that are easier to collect, transport, process and store. We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, femur, metatarsal, patella, rib and vertebral samples from five recently deceased untreated male cadavers and seven male human remains that were embalmed, buried for ∼ 1 month and then exhumed. The bodies had undergone different environmental conditions and were in various stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by an organic procedure was used. The utility of bone marrow and vitreous fluid including bone marrow and vitreous fluid that was transferred on FTA(®) cards and subjected to autosomal STR and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or absence of PCR inhibitors in DNA extracts was assessed using Plexor(®)HY. All samples were amplified using PowerPlex(®)21 and PowerPlexY(®)23 systems and analyzed using the AB3500 Genetic Analyzer and the GeneMapper(®) ID-X v.1.2 software. PCR inhibitors were consistently detected in bone marrow, muscle tissue, rib and vertebra samples. Amplifiable DNA was obtained in a majority of the samples analyzed. DNA recovery from 0.1g biological material was adequate for successful genotyping of most of the non-bone and bone samples. Complete DNA profiles were generated from bone marrow, femur, metatarsal and patella with 0.1 ng DNA template. Using 0.5 ng DNA template resulted in increased allele recovery and improved intra- and inter-locus peak balance.

Allele frequencies of 23 autosomal short tandem repeat loci in the Philippine population.

We characterized diversity and forensic descriptive parameters of 23 autosomal STR loci (CSF1PO, D13S317, D16S539, D5S818, D7S820, TPOX, D18S51, D21S11, D3S1358, D8S1179, FGA, TH01, vWA, D1S1656, D10S1248, D12S391, D2S441, D22S1045, D19S433, D2S1338, D6S1043, Penta D and Penta E) among 167 unrelated Filipinos. The most variable autosomal STR loci observed is Penta E (observed heterozygosity: 0.9222, match probability: 0.0167). Results reveal matching probability of 8.21×10(-28) for 23 autosomal STR loci. This dataset for the Philippine population may now be used in evaluating the weight of DNA evidence for forensic applications such as in human identification, parentage/kinship testing, and interpretation of DNA mixtures.

Complete mtDNA genomes of Filipino ethnolinguistic groups: a melting pot of recent and ancient lineages in the Asia-Pacific region.

The Philippines is a strategic point in the Asia-Pacific region for the study of human diversity, history and origins, as it is a cross-road for human migrations and consequently exhibits enormous ethnolinguistic diversity. Following on a previous in-depth study of Y-chromosome variation, here we provide new insights into the maternal genetic history of Filipino ethnolinguistic groups by surveying complete mitochondrial DNA (mtDNA) genomes from a total of 14 groups (11 groups in this study and 3 groups previously published) including previously published mtDNA hypervariable segment (HVS) data from Filipino regional center groups. Comparison of HVS data indicate genetic differences between ethnolinguistic and regional center groups. The complete mtDNA genomes of 14 ethnolinguistic groups reveal genetic aspects consistent with the Y-chromosome, namely: diversity and heterogeneity of groups, no support for a simple dichotomy between Negrito and non-Negrito groups, and different genetic affinities with Asia-Pacific groups that are both ancient and recent. Although some mtDNA haplogroups can be associated with the Austronesian expansion, there are others that associate with South Asia, Near Oceania and Australia that are consistent with a southern migration route for ethnolinguistic group ancestors into the Asia-Pacific, with a timeline that overlaps with the initial colonization of the Asia-Pacific region, the initial colonization of the Philippines and a possible separate post-colonization migration into the Philippine archipelago.

The Y-chromosome landscape of the Philippines: extensive heterogeneity and varying genetic affinities of Negrito and non-Negrito groups.

The Philippines exhibits a rich diversity of people, languages, and culture, including so-called 'Negrito' groups that have for long fascinated anthropologists, yet little is known about their genetic diversity. We report here, a survey of Y-chromosome variation in 390 individuals from 16 Filipino ethnolinguistic groups, including six Negrito groups, from across the archipelago. We find extreme diversity in the Y-chromosome lineages of Filipino groups with heterogeneity seen in both Negrito and non-Negrito groups, which does not support a simple dichotomy of Filipino groups as Negrito vs non-Negrito. Filipino non-recombining region of the human Y chromosome lineages reflect a chronology that extends from after the initial colonization of the Asia-Pacific region, to the time frame of the Austronesian expansion. Filipino groups appear to have diverse genetic affinities with different populations in the Asia-Pacific region. In particular, some Negrito groups are associated with indigenous Australians, with a potential time for the association ranging from the initial colonization of the region to more recent (after colonization) times. Overall, our results indicate extensive heterogeneity contributing to a complex genetic history for Filipino groups, with varying roles for migrations from outside the Philippines, genetic drift, and admixture among neighboring groups.

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.

Mapping human genetic diversity in Asia.

Asia harbors substantial cultural and linguistic diversity, but the geographic structure of genetic variation across the continent remains enigmatic. Here we report a large-scale survey of autosomal variation from a broad geographic sample of Asian human populations. Our results show that genetic ancestry is strongly correlated with linguistic affiliations as well as geography. Most populations show relatedness within ethnic/linguistic groups, despite prevalent gene flow among populations. More than 90% of East Asian (EA) haplotypes could be found in either Southeast Asian (SEA) or Central-South Asian (CSA) populations and show clinal structure with haplotype diversity decreasing from south to north. Furthermore, 50% of EA haplotypes were found in SEA only and 5% were found in CSA only, indicating that SEA was a major geographic source of EA populations.

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.

Fungal DNA challenge in human STR typing of bone samples.

The present study focuses on possible cross-reaction of fungal DNA with human STR primers that may affect subsequent forensic DNA analysis of forensic samples. Specificity of human STR markers namely HUMAMEL, HUMCSF1PO, D8S306, HUMTH01, HUMvWA, HUMFES/FPS, HUMF13A01, HUMDHFRP2, HUMFGA and HUMTPOX was tested using DNA of 24 different filamentous fungal isolates obtained from exhumed bone samples. The specificity of these ten STR markers for human DNA was demonstrated. Presence of non-human DNA in five bone samples analyzed did not alter scoring of detected alleles. Notably, amplification was inhibited in the presence of a high proportion of fungal DNA compared to human DNA (1000 ng: 1 ng) in DNA mixture experiments. The results of the present study underscore the importance of carefully analyzing the presence of non-human biological contaminants that may affect DNA typing of environmentally challenged forensic samples to avoid spurious data interpretation.

Identification of exhumed remains of fire tragedy victims using conventional methods and autosomal/Y-chromosomal short tandem repeat DNA profiling.

In a fire tragedy in Manila in December 1998, one of the worst tragic incidents which resulted in the reported death of 23 children, identity could not be established initially resulting in the burial of still unidentified bodies. Underscoring the importance of identifying each of the human remains, the bodies were exhumed 3 months after the tragedy. We describe here our work, which was the first national case handled by local laboratories wherein conventional and molecular-based techniques were successfully applied in forensic identification. The study reports analysis of DNA obtained from skeletal remains exposed to conditions of burning, burial, and exhumation. DNA typing methods using autosomal and Y-chromosomal short tandem repeat (Y-STR) markers reinforced postmortem examinations using conventional identification techniques. The strategy resulted in the identification of 18 out of the 21 human remains analyzed, overcoming challenges encountered due to the absence of established procedures for the recovery of mass disaster remains. There was incomplete antemortem information to match the postmortem data obtained from the remains of 3 female child victims. Two victims were readily identified due to the availability of antemortem tissues. In the absence of this biologic material, parentage testing was performed using reference blood samples collected from parents and relatives. Data on patrilineal lineage based on common Y-STR haplotypes augmented autosomal DNA typing, particularly in deficiency cases.

Identification of two fire victims by comparative nuclear DNA typing of skeletal remains and stored umbilical tissues.

We describe here our collaborative efforts in identifying 2 fatalities of a fire disaster by using a variety of identification techniques. Postmortem findings in both cases were reinforced using Short Tandem Repeat (STR) DNA technology to establish with a high degree of certainty the identities of 2 child victims. STR markers used in the present study include HUMAMEL, HUMCSFIPO, HUMTHO1, HUMvWA, HUMFES/FPS, HUMF13A01, HUMFOLP23, D8S3O6, HUMFGA, and HUMTPOX. Unambiguous identification was made possible through matching DNA profiles generated from skeletal remains with those from umbilical tissues. These tissues were kept by their mothers in accordance with a Philippine tradition and were submitted for DNA analysis. Of the DNA profiles generated from exhumed bone samples of 21 child victims, comparison with the genetic profiles of children A and B obtained from umbilical tissues showed consistent DNA matches with remains 1756 and 1758, respectively.