Genes

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18 pages, 4443 KiB

Open AccessArticle

Preliminary Study: DNA Transfer and Persistence on Non-Porous Surfaces Submerged in Spring Water

by Morgan L. Korzik, Josep De Alcaraz-Fossoul, Michael S. Adamowicz and David San Pietro

Genes 2023, 14(5), 1045; https://doi.org/10.3390/genes14051045 - 06 May 2023

Viewed by 2046

Submerged items are often thought to lack evidentiary value. However, previous studies have shown the ability to recover DNA from submerged porous items for upwards of six weeks. The crevices or interweaving fibers in porous items are thought to protect DNA from being [...] Read more.

Submerged items are often thought to lack evidentiary value. However, previous studies have shown the ability to recover DNA from submerged porous items for upwards of six weeks. The crevices or interweaving fibers in porous items are thought to protect DNA from being washed away. It is hypothesized that, because non-porous surfaces do not have the same traits that might aid in DNA retention, then DNA quantities and the number of donor alleles recovered would decrease over longer submersion periods. Additionally, it is hypothesized that DNA quantity and the number of alleles would be negatively affected by flow conditions. Neat saliva of known DNA quantity was applied to glass slides and exposed to stagnant and flowing spring water to observe the effects on both DNA quantity and STR detection. Results supported that DNA deposited onto glass and subsequently submerged in water experienced a decrease in DNA quantity over time, yet submersion did not have as strong of a negative effect on the detected amplification product. Additionally, an increase in DNA quantity and detected amplification product from designated blank slides (no initial DNA added) could indicate the possibility of DNA transfer. Full article

(This article belongs to the Special Issue Advances in Forensic Molecular Genetics)

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12 pages, 1175 KiB

Open AccessArticle

Evaluation and Verification of a microRNA Panel Using Quadratic Discriminant Analysis for the Classification of Human Body Fluids in DNA Extracts

by Ciara Rhodes, Carolyn Lewis, Kelsey Price, Anaya Valentine, Mary-Randall A. Creighton, Edward Boone and Sarah Seashols-Williams

Genes 2023, 14(5), 968; https://doi.org/10.3390/genes14050968 - 25 Apr 2023

Cited by 1 | Viewed by 3027

Abstract

There is significant interest in the use of miRNA analysis for forensic body fluid identification. Demonstrated co-extraction and detection in DNA extracts could make the use of miRNAs a more streamlined molecular body fluid identification method than other RNA-based methods. We previously reported [...] Read more.

There is significant interest in the use of miRNA analysis for forensic body fluid identification. Demonstrated co-extraction and detection in DNA extracts could make the use of miRNAs a more streamlined molecular body fluid identification method than other RNA-based methods. We previously reported a reverse transcription-quantitative PCR (RT-qPCR) panel of eight miRNAs that classified venous and menstrual blood, feces, urine, saliva, semen, and vaginal secretions using a quadratic discriminant analysis (QDA) model with 93% accuracy in RNA extracts. Herein, miRNA expression in DNA extracts from 50 donors of each body fluid were tested using the model. Initially, a classification rate of 87% was obtained, which increased to 92% when three additional miRNAs were added. Body fluid identification was found to be reliable across population samples of mixed ages, ethnicities, and sex, with 72–98% of the unknown samples classifying correctly. The model was then tested against compromised samples and over biological cycles, where classification accuracy varied, depending on the body fluid. In conclusion, we demonstrated the ability to classify body fluids using miRNA expression from DNA extracts, eliminating the need for RNA extraction, greatly reducing evidentiary sample consumption and processing time in forensic laboratories, but acknowledge that compromised semen and saliva samples can fail to classify properly, and mixed sample classification remains untested and may have limitations. Full article

(This article belongs to the Special Issue Advances in Forensic Molecular Genetics)

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23 pages, 2815 KiB

Open AccessArticle

Whole Mitochondrial Genome Detection and Analysis of Two- to Four-Generation Maternal Pedigrees Using a New Massively Parallel Sequencing Panel

by Dan Peng, Jiaojiao Geng, Jingyi Yang, Jiajun Liu, Nana Wang, Riga Wu and Hongyu Sun

Genes 2023, 14(4), 912; https://doi.org/10.3390/genes14040912 - 14 Apr 2023

Cited by 1 | Viewed by 1571

Abstract

Mitochondrial DNA (mtDNA) is an effective genetic marker in forensic practice, especially for aged bones and hair shafts. Detection of the whole mitochondrial genome (mtGenome) using traditional Sanger-type sequencing is laborious and time-consuming. Additionally, its ability to distinguish point heteroplasmy (PHP) and length [...] Read more.

Mitochondrial DNA (mtDNA) is an effective genetic marker in forensic practice, especially for aged bones and hair shafts. Detection of the whole mitochondrial genome (mtGenome) using traditional Sanger-type sequencing is laborious and time-consuming. Additionally, its ability to distinguish point heteroplasmy (PHP) and length heteroplasmy (LHP) is limited. The application of massively parallel sequencing in mtDNA detection helps researchers to study the mtGenome in-depth. The ForenSeq mtDNA Whole Genome Kit, which contains a total of 245 short amplicons, is one of the multiplex library preparation kits for the mtGenome. We used this system to detect the mtGenome in the blood samples and hair shafts of thirty-three individuals from eight two-generation pedigrees, one three-generation pedigree, and one four-generation pedigree. High-quality sequencing results were obtained. Ten unique mtGenome haplotypes were observed in the mothers from the ten pedigrees. A total of 26 PHPs were observed using the interpretation threshold of 6%. Eleven types of LHPs in six regions were evaluated in detail. When considering homoplasmic variants only, consistent mtGenome haplotypes were observed between the twice-sequenced libraries and between the blood and hair shafts from the same individual and among maternal relatives in the pedigrees. Four inherited PHPs were observed, and the remainder were de novo/disappearing PHPs in the pedigrees. Our results demonstrate the effective capability of the ForenSeq mtDNA Whole Genome Kit to generate the complete mtGenome in blood and hair shafts, as well as the complexity of mtDNA haplotype comparisons between different types of maternal relatives when heteroplasmy is considered. Full article

(This article belongs to the Special Issue Advances in Forensic Molecular Genetics)

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12 pages, 274 KiB

Open AccessArticle

The Analysis of Genetic Polymorphism on Mitochondrial Hypervariable Region III in Thai Population

by Jirat Vanichanukulyakit, Supakit Khacha-ananda, Tawachai Monum, Phatcharin Mahawong, Kittikhun Moophayak, Watsana Penkhrue, Taddaow Khumpook and Sorawat Thongsahuan

Genes 2023, 14(3), 682; https://doi.org/10.3390/genes14030682 - 09 Mar 2023

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Abstract

Mitochondrial DNA (mtDNA) analysis is a genetic marker for human identification, especially matrilineal inheritance. Hypervariable regions (HVR) I and II of mtDNA have been currently performed for human identification worldwide. Further examination of HVRIII has been conducted with the aim of enhancing the [...] Read more.

Mitochondrial DNA (mtDNA) analysis is a genetic marker for human identification, especially matrilineal inheritance. Hypervariable regions (HVR) I and II of mtDNA have been currently performed for human identification worldwide. Further examination of HVRIII has been conducted with the aim of enhancing the power of discrimination. The aim of this research is to provide informative data on the polymorphisms of HVRIII in the Thai population in order to establish a national database for human identification. Thai people who were unrelated through the maternal lineage were recruited for blood collections. The mtDNA was extracted by Chelex extraction, amplified by polymerase chain reaction, and analyzed using Sequencing Analysis Software. The most common mutation in HVRIII was base substitution, followed by deletion and insertion. We discovered 40 unique haplotypes, with haplotype 489C being the most frequent. The haplotype diversity, power of discrimination, and random match probability were 0.8014, 0.7987, and 0.2013, respectively. Five-CA repeats were the most frequently observed in nucleotide positions 514–523. Our database can be employed as supplementary markers in addition to nuclear deoxyribonucleic acid (DNA) markers in forensic investigations. Moreover, the data could potentially enhance genetic identification and anthropological genetics research in Thailand. Full article

(This article belongs to the Special Issue Advances in Forensic Molecular Genetics)

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23 pages, 6965 KiB

Open AccessArticle

Validation of Probabilistic Genotyping Software for Single Cell STR Analysis

by Kaitlin Huffman and Jack Ballantyne

Genes 2023, 14(3), 674; https://doi.org/10.3390/genes14030674 - 08 Mar 2023

Cited by 3 | Viewed by 1523

Abstract

Probabilistic genotyping (PG) and its associated software has greatly aided in forensic DNA mixture analysis, with it primarily being applied to mixed DNA profiles obtained from bulk cellular extracts. However, these software applications do not always result in probative information about the identity [...] Read more.

Probabilistic genotyping (PG) and its associated software has greatly aided in forensic DNA mixture analysis, with it primarily being applied to mixed DNA profiles obtained from bulk cellular extracts. However, these software applications do not always result in probative information about the identity of all donors to said mixtures/extracts. This is primarily due to mixture complexity caused by overlapping alleles and the presence of artifacts and minor donors. One way of reducing mixture complexity is to perform direct single cell subsampling of the bulk mixture prior to genotyping and interpretation. The analysis of low template DNA samples, including from single or few cells, has also benefited from the application of PG methods. With the application of PG, multiple cell subsamples originating from the same donor can be combined into a single analysis using the software replicate analysis function often resulting in full DNA profile donor information. In the present work, we demonstrate how two PG software systems, STRmix^TM and EuroForMix, were successfully validated for single or few cell applications. Full article

(This article belongs to the Special Issue Advances in Forensic Molecular Genetics)

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14 pages, 833 KiB

Open AccessArticle

Body Fluid Identification in Samples Collected after Intimate and Social Contact: A Comparison of Two mRNA Profiling Methods and the Additional Information Gained by cSNP Genotypes

by Helen Johannessen, Erin Hanson, Peter Gill, Cordula Haas, Erik Francisco Bergseth, Jack Ballantyne and Ane Elida Fonneløp

Genes 2023, 14(3), 636; https://doi.org/10.3390/genes14030636 - 03 Mar 2023

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Abstract

The ability to associate a contributor with a specific body fluid in a crime stain can aid casework investigation. The detection of body fluids combined with DNA analyses may supply essential information, but as the two tests are independent, they may not be [...] Read more.

The ability to associate a contributor with a specific body fluid in a crime stain can aid casework investigation. The detection of body fluids combined with DNA analyses may supply essential information, but as the two tests are independent, they may not be associated. Recently, the analysis of coding region SNPs (cSNPs) within the RNA transcript has been proven to be a promising method to face this challenge. In this study, we performed targeted RNA sequencing of 158 samples (boxershorts, fingernail swabs and penile swabs) collected from 12 couples at different time points post-intimate contact and after non-intimate contact, using the Ion S5™ System and BFID-cSNP-6F assay. The aim of the study was to compare the performance of the MPS and CE methods in the detection of mRNA markers, and to associate body fluids with contributors by their cSNP genotypes. The results of the study show a lower success rate in the detection of vaginal mucosa by the MPS compared to the CE method. However, the additional information obtained with the cSNP genotypes could successfully associate body fluids with contributors in most cases. Full article

(This article belongs to the Special Issue Advances in Forensic Molecular Genetics)

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16 pages, 1042 KiB

Open AccessArticle

Open-Access Worldwide Population STR Database Constructed Using High-Coverage Massively Parallel Sequencing Data Obtained from the 1000 Genomes Project

by Tamara Soledad Frontanilla, Guilherme Valle-Silva, Jesus Ayala and Celso Teixeira Mendes-Junior

Genes 2022, 13(12), 2205; https://doi.org/10.3390/genes13122205 - 24 Nov 2022

Cited by 2 | Viewed by 1901

Abstract

Achieving accurate STR genotyping by using next-generation sequencing data has been challenging. To provide the forensic genetics community with a reliable open-access STR database, we conducted a comprehensive genotyping analysis of a set of STRs of broad forensic interest obtained from 1000 Genome [...] Read more.

Achieving accurate STR genotyping by using next-generation sequencing data has been challenging. To provide the forensic genetics community with a reliable open-access STR database, we conducted a comprehensive genotyping analysis of a set of STRs of broad forensic interest obtained from 1000 Genome populations. We analyzed 22 STR markers using files of the high-coverage dataset of Phase 3 of the 1000 Genomes Project. We used HipSTR to call genotypes from 2504 samples obtained from 26 populations. We were not able to detect the D21S11 marker. The Hardy-Weinberg equilibrium analysis coupled with a comprehensive analysis of allele frequencies revealed that HipSTR was not able to identify longer alleles, which resulted in heterozygote deficiency. Nevertheless, AMOVA, a clustering analysis that uses STRUCTURE, and a Principal Coordinates Analysis showed a clear-cut separation between the four major ancestries sampled by the 1000 Genomes Consortium. Except for larger Penta D and Penta E alleles, and two very small Penta D alleles (2.2 and 3.2) usually observed in African populations, our analyses revealed that allele frequencies and genotypes offered as an open-access database are consistent and reliable. Full article

(This article belongs to the Special Issue Advances in Forensic Molecular Genetics)

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20 pages, 4227 KiB

Open AccessArticle

Routine Mitogenome MPS Analysis from 1 and 5 mm of Rootless Human Hair

by Lauren C. Canale, Jennifer A. McElhoe, Gloria Dimick, Katherine M. DeHeer, Jason Beckert and Mitchell M. Holland

Genes 2022, 13(11), 2144; https://doi.org/10.3390/genes13112144 - 18 Nov 2022

Cited by 1 | Viewed by 2766

Abstract

While hair shafts are a common evidence type in forensic cases, they are often excluded from DNA analysis due to their limited DNA quantity and quality. Mitochondrial (mt) DNA sequencing is the method of choice when working with rootless hair shaft fragments due [...] Read more.

While hair shafts are a common evidence type in forensic cases, they are often excluded from DNA analysis due to their limited DNA quantity and quality. Mitochondrial (mt) DNA sequencing is the method of choice when working with rootless hair shaft fragments due to the elevated copy number of mtDNA and the highly degraded nature of nuclear (n) DNA. Using massively parallel sequencing (MPS) of the mitochondrial (mito) genome, we studied the impact of hair age (time since collection) and physical characteristics (hair diameter, medullary structure, and length of hair tested) on mtDNA recovery and MPS data quality. Hair shaft cuttings of 1 and 5 mm from hairs less than five years to 46 years of age from 60 donors were characterized microscopically. Mitogenome sequences were generated using the Promega PowerSeq^TM Whole Mito System prototype kit and the Illumina MiSeq instrument. Reportable mitogenome sequences were obtained from all hairs up to 27 years of age (37 donors), with at least 98% of the mitogenome reported for more than 94% of the 74 hair samples analyzed; the minimum reported sequence was 88%. Furthermore, data from the 1 and 5 mm replicates gave concordant haplotypes. As expected, mtDNA yield decreased, mtDNA degradation increased, and mitogenome MPS data quality declined as the age of the hair increased. Hair diameter and medullary structure had minimal impact on yield and data quality. Our findings support that MPS is a robust and reliable method for routinely generating mitogenome sequences from 1 and 5 mm hair shaft samples up to 27 years of age, which is of interest to the forensic community, biological anthropologists, and medical geneticists. Full article

(This article belongs to the Special Issue Advances in Forensic Molecular Genetics)

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