Microsatellite Research Articles

Searching for alternative high DNA-yielding bone types for DNA analysis of aged skeletal remains

The petrous bone contains significantly higher amounts of DNA than any other human bone. Because of highly destructive sampling and because it is not always part of the recovered remains, the need for alternative sources of DNA is important. To identify additional optimal bone types, petrous bones were compared to femurs, tali, and calcanei sampled from 66 adult skeletons from two distinct modern-era Christian cemeteries. An extraction method employing full demineralization was used to obtain DNA, real-time PCR quantification to ascertain DNA quantity and degradation, and a commercial forensic short tandem repeats (STR) PCR amplification kit to determine genetic profiles. Statistical analysis was performed to explore the differences in DNA yield, DNA degradation, and success of STR amplification. A systematic studies exploring intra-skeletal variability in DNA preservation including various excavation sites differing by time period and geographical position are rare, and the second part of the investigation was based on a comparison of both archaeological sites, which allowed us to compare the effect of different post-mortem intervals and environmental conditions on DNA preservation. The older burial site in Črnomelj was active between the 13th and 18th century, whereas the more recent Polje burial was in use from the 16th to 19th century, creating different temporal and geographical environments. Results for the Črnomelj burial site revealed that the petrous bone outperformed all other bone types studied, except the calcaneus. At the Polje archeological site calcanei, tali, and femurs yielded the same STR typing success as petrous bones. The results obtained highlight the importance of careful bone sample selection for DNA analysis of aged skeletal remains. In addition to petrous bones, calcanei were found to be an alternative source of DNA when older burial sites are investigated. When more recent burial sites are processed, calcanei, tali, and femurs should be sampled besides petrous bones, not only because they exhibited good performance, but also because of easier sampling and easier grinding in the case of trabecular bones. This study contributes valuable insights into the potential use of various skeletal types as a source of DNA for investigation of aged skeletal remains, and it offers practical implications for forensic and archaeological investigations.

Estimating genetic diversity among selected wild grapevine genotypes from Southern Turkey by simple sequence repeat (SSR) and inter-Primer Binding Site(iPBS) markers

Estimating genetic diversity among selected wild grapevine genotypes from Southern Turkey by simple sequence repeat (SSR) and inter-Primer Binding Site(iPBS) markers

Clinical application of whole genome sequencing in young onset dementia: challenges and opportunities.

Young onset dementia (YOD) by its nature is difficult to diagnose. Despite involvement of multidisciplinary neurogenetics services, patients with YOD and their families face significant diagnostic delays. Genetic testing for people with YOD currently involves a staggered, iterative approach. There is currently no optimal single genetic investigation that simultaneously identifies the different genetic variants resulting in YOD. This review discusses the advances in clinical genomic testing for people with YOD. Whole genome sequencing (WGS) can be employed as a 'one stop shop' genomic test for YOD. In addition to single nucleotide variants, WGS can reliably detect structural variants, short tandem repeat expansions, mitochondrial genetic variants as well as capture single nucleotide polymorphisms for the calculation of polygenic risk scores. WGS, when used as the initial genetic test, can enhance the likelihood of a precision diagnosis and curtail the time taken to reach this. Finding a clinical diagnosis using WGS can reduce invasive and expensive investigations and could be cost effective. These advances need to be balanced against the limitations of the technology and the genetic counseling needs for these vulnerable patients and their families.

Molecular diversity of rice germplasms from Thailand characterized by different pericarp colors using Inter Simple Sequence Repeat (ISSR) markers

Abstract. Saengprajak J, Phetsom J, Sangdee A, Atichart P, Theerakulpisut P. 2024. Molecular diversity of rice germplasms from Thailand characterized by different pericarp colors using Inter Simple Sequence Repeat (ISSR) markers. Biodiversitas 25: 2839-2848. Studying genetic diversity in rice using molecular markers is necessary for varietal identification, better understanding of relationships and evolution, and conserving genetic resources. This study aimed to evaluate the genetic diversity of rice germplasms from Thailand characterized by different pericarp colors using ISSR markers. Thirty-two white and colored rice genotypes were assessed using 16 selected ISSR markers, which generated 179 alleles ranging from 210 to 1,374 bp, averaging 11.19 alleles per locus. The polymorphism percentage ranged from 40% to 100%, averaging 80%. The genetic similarity coefficient ranged from 0.458 to 0.855, while PIC values varied between 0.15 and 0.45, averaging 0.31. ISSR markers show a high Resolving Power (RP) of 12.15 and a Marker Index (MI) of 3.20, indicating their significant discriminatory power and effectiveness in detecting genetic diversity. The dendrogram grouped genotypes into five clusters: Cluster I included 5 red pericarp genotypes, Cluster II had 6 purple-black pericarp genotypes, Cluster III had 2 mixed white and purple-black pericarp genotypes, Cluster IV included 18 white pericarp genotypes, and Cluster V comprised 1 white pericarp genotype. PCA results corresponded to seed coat traits, forming eight grouping patterns similar to dendrogram clusters. These findings show that ISSR markers effectively assess genetic diversity and relationships in rice germplasms, providing valuable information for managing germplasm resources, developing variety-specific markers, and supporting rice breeding programs.

Analysis of chemical and genetic variability in wild hop (Humulus lupulus L.) populations of Kosovo.

Hops is an economically important species due to its diverse secondary metabolites and extensive use in the brewing and medicinal industries. Although hops is widely distributed in Kosovo, the chemical composition of its essential oils and genetic variability of wild populations remain understudied. Therefore, this study aimed to evaluate the chemical and genetic variability of Kosovo's wild hop population using essential oil constituents and microsatellite (simple sequence repeat - SSR) markers. Female hop inflorescences were collected from 21 wild populations in Kosovo. Essential oils were extracted from the dried plant material using a Clevenger apparatus. Chemical composition of the essential oils was analysed using GC-FID-MS. DNA was extracted from dried leaves, and 15 SSR markers were used for fragment analysis. The main constituents of the essential oil were myrcene, α-humulene, (E)-β-farnesene, α-selinene, β-selinene, and E-caryophyllene. Statistical analyses based on chemical composition of essential oils and SSR markers highlighted the low variability among populations and high variability within populations. These findings provide valuable insights for developing strategies for potential use and conservation of wild hop populations in Kosovo, laying the groundwork for future research and comparison with commercial cultivars to assess their breeding potential.

Establishment of Novel Simple Sequence Repeat (SSR) Markers from Chimonanthus praecox Transcriptome Data and Their Application in the Identification of Varieties.

Chimonanthus praecox, a member of the Calycanthaceae family, is a unique, traditional, and famous flowering economic tree species in China. Despite the existence of several varieties, only a few cultivars have been formally named. Currently, expression sequence tag-simple sequence repeat (EST-SSR) markers are extensively used to identify different species and varieties; a large number of microsatellites can be identified from transcriptome databases. A total of 162,638 unigenes were assembled using RNA-seq; 82,778 unigenes were annotated using the Nr, Nt, Swiss-Prot, Pfam, GO, KOG, and KEGG databases. In total, 13,556 SSR loci were detected from 11,691 unigenes, with trinucleotide repeat motifs being the most abundant among the six repeat motifs. To develop the markers, 64,440 pairs of SSR primers with polymorphism potential were designed, and 75 pairs of primers were randomly selected for amplification. Among these markers, seven pairs produced amplified fragments of the expected size with high polymorphism. Using these markers, 12 C. praecox varieties were clustered into two monophyletic clades. Microsatellites in the transcriptome of C. praecox exhibit rich types, strong specificity, and great polymorphism potential. These EST-SSR markers serve as molecular technical methods for identifying different varieties of C. praecox and facilitate the exploration of a large number of candidate genes associated with important traits.

Hatcheries maintain high genetic variation but show shifts in genetic structure of progenies of five endangered riffleshell species (Epioblasma) (Bivalvia: Unionidae) of the eastern United States

Abstract We assessed genetic variation at mitochondrial DNA (mtDNA) and nuclear DNA microsatellites for progeny (i.e., juveniles produced at a hatchery) of five endangered Epioblasma species reared at three hatcheries in the eastern United States. The progeny of Epioblasma aureola, Epioblasma brevidens, Epioblasma obliquata and Epioblasma triquetra showed no loss of mtDNA haplotype diversity relative to broodstock (i.e., wild collected gravid females used to produce juveniles) and wildstock (i.e., individuals sampled in the wild to assess baseline genetic diversity), while progeny of Epioblasma capsaeformis showed no loss relative to broodstock but an ~50% loss relative to wildstock. At DNA microsatellites, mean expected heterozygosities (He) were maintained in wildstock, broodstock and progeny, with the lowest values observed in E. aureola and E. triquetra. Among progeny, values of He and allelic richness (A) at times exceeded those observed in the wildstock and broodstock. Hence, no loss of genetic variation at DNA microsatellites was observed in progeny among species. We documented multiple paternity in progeny of E. aureola, E. capsaeformis and E. obliquata and in part attribute their high He and A to fertilization of broodstock females by multiple males in the wild. We observed significant divergence in FST and D values between progeny to wildstock and progeny to broodstock. Most pairwise comparisons for E. brevidens and E. capsaeformis were significantly diverged, and for E. obliquata, divergence was low but also significantly different, and only for E. aureola was it low and nonsignificant. Our results showed that population genetic structure can develop quickly between progeny and their progenitors in the first generation (F1) of offspring produced from a set of parents and that genetic diversity at mtDNA and nuclear DNA microsatellites was generally maintained in progeny of endangered Epioblasma species reared at three mussel hatcheries utilizing current propagation practices.

Development and characterization of a novel intergeneric hybrid grouper (Cromileptes altivelis ♀ × Epinephelus lanceolatus ♂)

Distant hybridization often gives rise to offspring with hybrid vigor in plants and animals, and this method has also shown great potential in aquaculture. Grouper, an economically important marine species, has witnessed rapid growth in its related industry. To cultivate a new grouper variety with accelerated growth, superior meat quality, and high economic value, we successfully obtained fertilized eggs through artificial insemination, using humpback grouper (Cromileptes altivelis) as the maternal parent and giant grouper (Epinephelus lanceolatus) as the paternal parent. The hybrid groupers were successfully hatched and reared for up to 14 months. This study conducted comprehensive comparative analyses between the hybrid offspring and the parental species, focusing on embryo development, growth rate, morphological characteristics, chromosome karyotype and muscle nutrient composition. The hybrid grouper embryos completed their entire development, from fertilization to hatching, in an average duration of 20 h and 37 min, under controlled conditions of 28.5 ± 0.5 °C temperature and 28 part per thousand (ppt) salinity. Remarkably, the hybrid groupers exhibited significantly faster growth than humpback groupers, with an absolute weight growth rate 1.6 times higher. Additionally, the hybrid groupers displayed a 4.7% increase in meat yield compared to giant groupers. In terms of morphology, cluster analysis and principal component analysis were used to analyze the morphological framework data of the three grouper species, revealing that the hybrid groupers were more similar to giant groupers in their morphological framework. Chromosome karyotype and simple sequence repeats (SSR) analysis confirmed that the hybrid groupers have a total of 48 chromosomes, inheriting one set of chromosomes from each parent. This study provides a foundation for grouper hybrid breeding, and the hybrid groupers obtained in this research have the potential to become excellent commercial varieties.

Prevalence of Transcription Factor 4 Gene Triplet Repeat Expansion Associated with Fuchs’ Endothelial Corneal Dystrophy in the United States and Global Populations

ObjectiveAn intronic CTG triplet repeat expansion in the transcription factor 4 gene (TCF4) gene (CTG18.1) confers significant risk for the development of Fuchs’ endothelial corneal dystrophy (FECD). The objective of this study was to conduct an unbiased survey of the CTG18.1 repeat expansion allele frequencies in a multi-ethnic, population-based cohort from the United States and in global populations. DesignCross-sectional study SubjectsDallas Heart Study (DHS) cohort including 1,599 African Americans (AAs), 1,028 European Americans (EAs), and 458 Latinos; 2,500 individuals from the 1000 Genomes Project (1KGP) sampled from 26 populations across 5 continents. MethodsWe genotyped the CTG18.1 short tandem repeat in DHS using targeted polymerase chain reaction amplification followed by fragment analysis. We also inferred the CTG18.1 repeat genotype based on short-read whole-genome sequencing in 1KGP using the computational tool ExpansionHunter. Main Outcome MeasuresThe prevalence of an expanded CTG18.1 allele with ≥40 repeats was determined in U.S. and global populations. ResultsThe carrier rates of the expanded allele were 3.1%, 8.1%, and 3.3% in AAs, EAs, and Latinos, respectively, in the DHS, and 2.7%, 9.5%, 5.2%, 7.2%, and 5.2% in the African (AFR), European (EUR), East Asian (EAS), South Asian (SAS), and admixed American (AMR) continental populations, respectively, in the 1KGP. The distributions of the CTG18.1 repeat in DHS and in 1KGP are similar. The median repeat length was ∼17 with the interquartile range (IQR) between 12 and 23 in the DHS populations. The median repeat length was ∼19 in all the 1KGP populations with the IQR between 13 and 26. The highest prevalence of the expanded allele carriers ranging from 12.1% to 12.5% was observed in some EUR and AMR subpopulations. The frequency of expanded alleles carriers was absent or low (0 - 1.9%) in subpopulations of East Africa but was present at 6.2% in a Kenyan subpopulation in West Africa. ConclusionsThe TCF4 repeat expansion is most prevalent in people of European ancestry and least in African ancestry, which is consistent with FECD prevalence. The expanded TCF4 CTG18.1 allele is the most common disease-causing short tandem repeat in humans with worldwide implications for corneal disease.

Haplotype-based Analysis of 6q24-25 for Association with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a multisystem inflammatory disorder. Family history of RA is an important risk factor as it is strongly linked with the inherited HLA-DR4 (most specifically DR0401 and 0404). The aim of this study is to conduct the haplotype-based analysis of 6q24-25 and evaluate its association with RA. Case-control study which included all patients attending outpatient department (OPD) at Sardar Patel Medical College, Bikaner, Rajasthan and volunteers (only for blood samples). Blood samples of patients were collected. As per inclusion and exclusion criteria, a total of 103 subjects lacking history of disease were included under control group, while 48 cases were recruited as study group. Any significant departure of genotype distribution is evaluated using Hardy-Weinberg equilibrium by Chi-squared test. Case-control association was done using data from 151 genomic deoxyribonucleic acid (DNA) samples, which were allele typed. RA is significantly associated with >305bp (the longer allele of D6S1053 corresponding to 11 tetramer (GATA) repeats. Differences in individual allele frequency within the control population and RA cases were observed which shows the bimodal distribution of the 10 alleles of D6S1053 short tandem repeat (STR) marker observed in the cohort tested by us. No significant association with the risk for RA is shown by the allele for D6S1053 and the mutant allele 118G. Similarly, OPRM1 gene's haplotype frequency for rs1799972 for D6S1053 allele has not shown any added risk with the wild allele 17C compared to controls. The polymorphism showed that 17T depicted a higher odds ratio of 1.3 with an associated risk of 1.15 in the presence of longer allele of DS61053. Significance observed between short allele and RA was lost when haplotype analysis for the two genes were taken together. There was no difference observed between the short or long allele and 17T/C while there was a significant difference observed when haplotype frequencies were compared with alleles of A118G and the long and short allele of DS61053. We concluded that the short allele of µ-opioid receptor (MOR) gene offered a clear protection from a risk of getting RA.

Performance Characteristics of Next-Generation Sequencing–Based Engraftment Monitoring and Microchimerism Detection in Allogeneic Hematopoietic Cell Transplantation: A Practical Approach for Clinical Assay Validation

Chimerism analysis by next-generation sequencing (NGS) is an emerging method for engraftment monitoring after allogeneic hematopoietic cell transplantation. A high-sensitivity method is required for the detection of microchimerism (<1% chimerism), which may have clinical utility in early relapse detection, allograft monitoring in organ transplantation, and other allogeneic cellular therapies (such as microtransplantations). As more clinical laboratories adopt this method, a thorough assessment of performance is needed. This study evaluated one such NGS-based assay that uses both single-nucleotide polymorphisms and insertions/deletions as genetic markers. An assessment of accuracy, linearity, sensitivity, and reproducibility was performed. Analytical sensitivity was 0.2% donor for single donor and 0.5% donors for double donors. The assay showed a high degree of reproducibility over a full range of chimerism. Comparison to short-tandem-repeat (STR) PCR showed high concordance; yet <5% chimerism was consistently detected by NGS, but not by STR-PCR. Comparison to real-time quantitative PCR showedhigh concordance, but with lower correlation in the midrange (40% to 60% chimerism). Overall, the assay showed consistent performance with high sensitivity and accuracy compared with STR-PCR and real-time quantitative PCR across a full range of chimerism in the setting of single-donor and multidonor transplantations. In addition, criteria for quality metrics were established for sequencing performance and data analysis and considerations made for clinical laboratory validation of NGS-based chimerism assay andanalysis software.

Association Mapping of Seed Coat Color Characteristics for Near-Isogenic Lines of Colored Waxy Maize Using Simple Sequence Repeat Markers.

Waxy maize is mainly cultivated in South Korea for the production of food and snacks, and colored maize with increased anthocyanin content is used in the production of functional foods and medicinal products. Association mapping analysis (AMA) is supported as the preferred method for identifying genetic markers associated with complex traits. Our study aimed to identify molecular markers associated with two anthocyanin content and six seed coat color traits in near-isogenic lines (NILs) of colored waxy maize assessed through AMA. We performed AMA for 285 SSR loci and two anthocyanin content and six seed coat color traits in 10 NILs of colored waxy maize. In the analysis of population structure and cluster formation, the two parental lines (HW3, HW9) of "Mibaek 2ho" variety waxy maize and the 10 NILs were clearly divided into two groups, with each group containing one of the two parental inbred lines. In the AMA, 62 SSR markers were associated with two seed anthocyanin content and six seed coat color traits in the 10 NILs. All the anthocyanin content and seed coat color traits were associated with SSR markers, ranging from 2 to 12 SSR markers per characteristic. The 12 SSR markers were together associated with both of the two anthocyanin content (kuromanin and peonidin) traits. Our current results demonstrate the effectiveness of SSR analysis for the examination of genetic diversity, relationships, and population structure and AMA in 10 NILs of colored waxy maize and the two parental lines of the "Mibaek 2ho" variety waxy maize.

Genetic diversity and population structure of an insect-pollinated and bird-dispersed dioecious tree Magnolia kwangsiensis in a fragmented karst forest landscape.

This study combined population genetics and parentage analysis to obtain foundational data for the conservation of Magnolia kwangsiensis. M. kwangsiensis is a Class I tree species that occurs in two disjunct regions in a biodiversity hotspot in southwest China. We assessed the genetic diversity and structure of this species across its distribution range to support its conservation management. Genetic diversity and population structure of 529 individuals sampled from 14 populations were investigated using seven nuclear simple sequence repeat (nSSR) markers and three chloroplast DNA (cpDNA) fragments. Parentage analysis was used to evaluate the pollen and seed dispersal distances. The nSSR marker analysis revealed a high genetic diversity in M. kwangsiensis, with an average observed (Ho) and expected heterozygosities (He) of 0.726 and 0.687, respectively. The mean and maximum pollen and seed dispersal distances were 66.4 and 95.7 m and 535.4 and 553.8 m, respectively. Our data revealed two distinct genetic groups, consistent with the disjunct geographical distribution of the M. kwangsiensis populations. Both pollen and seed dispersal movements help maintain genetic connectivity among M. kwangsiensis populations, contributing to high levels of genetic diversity. Both genetically differentiated groups corresponding to the two disjunct regions should be recognized as separate conservation units.

Enhancing Clinicopathological Diagnosis of Hydatidiform Mole Through the Combined Application of Histomorphologic Analysis, Immunohistochemical Analysis of p57 Expression, and Short Tandem Repeat Typing Method

This study aims to assess the early histomorphologic characteristics and investigate the role of the p57kip2 protein combined with STR genotyping for pathological diagnosis and typing of the hydatidiform mole (HM). A total of 73 induced abortion tissues were collected for pathological evaluation, including 14 cases with partial HM (PHM), 7 cases with complete HM (CHM), and 52 cases with non-molar pregnancies. Histopathological examination of moles was conducted using hematoxylin and eosin staining. DNA extraction from paraffin sections was performed using Fe3O4 nano-magnetic beads. Molecular diagnosis was performed using STR genotyping. Immunohistochemical analysis was used to determine the distribution and expression level of p57kip2 protein in HM. Significant differences were observed in the morphological indices of villous edema, cistern formation, trophoblastic inclusions, and trophoblastic hyperplasia between the PHM and CHM groups (P &lt; 0.05). The central cistern formation and the trophoblast inclusion showed a significant difference between the HM and non-molar pregnancy (P &lt; 0.05). Moreover, our findings revealed that p57kip2 expression contributed to distinguishing CHM from PHM. However, it could not distinguish PHM from non-mole pregnancy. Furthermore, the results of STR genotyping were consistent with pathological typing. In conclusion, the integration of pathomorphology, immunohistochemical staining, and molecular diagnostics holds great value for the diagnosis and classification of HM.

Genetic Features of the Bream Abramis brama Population from the Lake System of Sebezhsky National Park

The genetic variability of microsatellite DNA has been studied for the first time for five loci in the common bream Abramis brama (L.) from lakes Sebezhskoye and Necheritsa of the Sebezhsky National Park lake system (Pskov oblast). The results demonstrate the uniformity of estimates of the genetic variability of the bream in the lake system. Estimates of the allelic diversity of microsatellite loci and the expected heterozygosity of local bream groups do not differ significantly. The total genetic differentiation of bream is θ = 0.004, 95% CI (–0.01; 0.01), and is statistically insignificant. The Bayesian analysis of the studied multilocus genotypes has not revealed any population–genetic structure. The results indicate a high level of gene flow between local bream groupings and make it possible to assume the presence of a genetically unified panmixed population in the Sebezhsky National Park lake system.

Assessment of Genetic Diversity and Relatedness of Local Banana (Musa spp.) Cultivars in Malaysia Using Simple Sequence Repeat (SSR) Markers

Banana (Musa spp.) is an economically important crop widely consumed all over the world. Understanding the genetic diversity and relatedness between various banana cultivars is important to continuously improve its fruit characteristics, nutritional value, and disease resistance. This study aimed to develop a DNA fingerprinting panel using polymorphic simple sequence repeat (SSR) markers for the genetic characterisation of 11 banana cultivars in Malaysia. A total of 10 polymorphic SSR markers were developed into a multiplex PCR reaction and capillary electrophoresis to uniquely profile our collection of banana cultivars. The developed fingerprinting panel successfully amplified a total of 95 alleles, with 6 to 13 alleles per SSR marker. The average SSR marker polymorphic information content (PIC) value is 0.812, indicating the informativeness of the panel. Analysis of molecular variance (AMOVA) shows that 97% of the molecular variance from our banana collection is due to inter-variety genetic diversity, while the remaining 3% is due to intra-variety genetic diversity. A population structure analysis groups our collection of banana varieties according to the presence of at least one M. balbisiana (B) genome in their genetic makeup. The Cavendish variety, however, showed a distinct structure compared to the other cultivars. This SSR fingerprinting panel provides valuable insights into the genetic diversity and relatedness between banana cultivars in Malaysia. It has the potential to assist future banana breeding initiatives and serve as an effective quality control measure for verifying varieties in a tissue culture facility involved in banana planting materials production.

Genetic structure and gene flow among populations of willow (Salix species)

The newly developed Salix hybrids were assessed for genetic diversity using ISSR (Inter Simple Sequence Repeats) molecular markers. The results revealed 88.08 polymorphism, 0.53 polymorphism information content, 4.70 effective multiplex ratio, 2.54 marker index, and 7.18 resolving power from the amplified primers. Clones of the Salix matsudana (MAT) population recorded a maximum mean number of effective alleles (1.42), Shannon information index (0.38), gene diversity (0.25), and percentage of polymorphic loci (82.24 %). The AMOVA (Analysis of molecular variance) results revealed a moderate genetic divergence with a genetic variation of 88 per cent within populations and 12 per cent among the populations. The highest value of total diversity (0.28) and gene diversity (0.24) was recorded between S. matsudana (MAT) and S. tetrasperma (TETRA) populations, while there was more gene flow between S. matsudana (MAT) and S. tetrasperma (TETRA) populations (5.86). The clustering pattern obtained through STRUCTURE software showed two main gene pools and a mixture of 6–7 gene pools to confirm the hybrids based on ISSR marker data. S. matsudana and S. tetrasperma clones showed the highest gene diversity due to their proximity from their original locations, resulting in the least amount of gene exchange among populations. Molecular characterization using ISSR markers was extremely useful for studying the genetic diversity among the willow hybrids and high genetic variability should be utilized for breeding purposes in the future.

Discordance of Penta D x.4 microvariant alleles results between three capillary electrophoresis and one massively parallel sequencing short tandem repeat kits

Forensic Biology is contingent upon matching DNA profiles between a crime sample and a reference sample. There are several capillary electrophoresis kits available to generate a short tandem repeat (STR) profile from DNA samples, while newer methods using massively parallel sequencing are slowly being implemented in forensic laboratories worldwide. During evaluation of a newer capillary electrophoresis kit, Applied Biosystems™ VeriFiler™ Plus, a discordance was observed in the Penta D locus. The previous kit, Promega PowerPlex 21® System produced a 13.4,14 genotype, whilst VeriFiler™ Plus produced a 14,14 genotype. An expanded investigation into Penta D microvariant alleles revealed that multiple discordances were observed for DNA profiles containing larger x.4 variants. There was full concordance between PowerPlex® 21 and QIAGEN Investigator® 26plex, however discordances were observed between VeriFiler™ Plus and the other three kits tested, including the massively parallel sequencing kit, Verogen ForenSeq® MainstAY. Notably, four of these discordances resulted in null alleles with the VeriFiler™ Plus kit. A review of the Penta D DNA sequences in MainstAY revealed fully concordant microvariant alleles involved deletions within the repeat region, whilst variability in the discordances observed were dependent on the location of the variation outside the repeat region and the analysis method used. Variations observed within the 5’ flanking region produced the same allele designation across all capillary electrophoresis kits. However, deletions within the 3’ region either produced a null allele for VeriFiler™ Plus where the deletion is thought to overlap the primer binding site, or microvariant alleles for the PowerPlex® 21 and Investigator 26plex kits, which produced longer Penta D amplicons. The discovery of these variations in the Penta D flanking sequences is informative as it increases the awareness of Penta D discordances between different kit chemistries in nominated reference DNA profile comparisons and DNA database searching and matching alike, and provides support for this phenomenon when providing evidence as to the admissibility of such results in trial proceedings.

Intrabreed variability of Landrace animals based on microsatellite DNA polymorphism

Intrabreed variability of Landrace animals based on microsatellite DNA polymorphism

Genome-wide detection of somatic mosaicism at short tandem repeats.

Somatic mosaicism has been implicated in several developmental disorders, cancers, and other diseases. Short tandem repeats (STRs) consist of repeated sequences of 1-6bp and comprise >1 million loci in the human genome. Somatic mosaicism at STRs is known to play a key role in the pathogenicity of loci implicated in repeat expansion disorders and is highly prevalent in cancers exhibiting microsatellite instability. While a variety of tools have been developed to genotype germline variation at STRs, a method for systematically identifying mosaic STRs is lacking. We introduce prancSTR, a novel method for detecting mosaic STRs from individual high-throughput sequencing datasets. prancSTR is designed to detect loci characterized by a single high-frequency mosaic allele, but can also detect loci with multiple mosaic alleles. Unlike many existing mosaicism detection methods for other variant types, prancSTR does not require a matched control sample as input. We show that prancSTR accurately identifies mosaic STRs in simulated data, demonstrate its feasibility by identifying candidate mosaic STRs in Illumina whole genome sequencing data derived from lymphoblastoid cell lines for individuals sequenced by the 1000 Genomes Project, and evaluate the use of prancSTR on Element and PacBio data. In addition to prancSTR, we present simTR, a novel simulation framework which simulates raw sequencing reads with realistic error profiles at STRs. prancSTR and simTR are freely available at https://github.com/gymrek-lab/trtools. Detailed documentation is available at https://trtools.readthedocs.io/.