RESUMO
Macrohaplotype combines multiple types of phased DNA variants, increasing forensic discrimination power. High-quality long-sequencing reads, for example, PacBio HiFi reads, provide data to detect macrohaplotypes in multiploidy and DNA mixtures. However, the bioinformatics tools for detecting macrohaplotypes are lacking. In this study, we developed a bioinformatics software, MacroHapCaller, in which targeted loci (i.e., short TRs [STRs], single nucleotide polymorphisms, and insertion and deletions) are genotyped and combined with novel algorithms to call macrohaplotypes from long reads. MacroHapCaller uses physical phasing (i.e., read-backed phasing) to identify macrohaplotypes, and thus it can detect multi-allelic macrohaplotypes for a given sample. MacroHapCaller was validated with data generated from our designed targeted PacBio HiFi sequencing pipeline, which sequenced â¼8-kb amplicon regions harboring 20 core forensic STR loci in human benchmark samples HG002 and HG003. MacroHapCaller also was validated in whole-genome long-read sequencing data. Robust and accurate genotyping and phased macrohaplotypes were obtained with MacroHapCaller compared with the known ground truth. MacroHapCaller achieved a higher or consistent genotyping accuracy and faster speed than existing tools HipSTR and DeepVar. MacroHapCaller enables efficient macrohaplotype analysis from high-throughput sequencing data and supports applications using discriminating macrohaplotypes.
Assuntos
Haplótipos , Sequenciamento de Nucleotídeos em Larga Escala , Polimorfismo de Nucleotídeo Único , Poliploidia , Análise de Sequência de DNA , Software , Humanos , Análise de Sequência de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Algoritmos , Biologia Computacional/métodos , DNA/genética , DNA/análise , Repetições de Microssatélites/genética , Genética Forense/métodos , Técnicas de Genotipagem/métodosRESUMO
Despite recent advances in the research related to air pollution and associated adverse cardiovascular events, the combined effects of air pollution, climate change, and SARS-CoV-2 infection on cardiovascular health need to be researched further. This Commentary addresses their impacts on cardiovascular health in the approximately 25 million people with a severe form of inherited hypercholesterolemia, called familial hypercholesterolemia (FH). The arterial endothelium in these individuals is potentially under multiple attacks caused by particles of both endogenous and exogenous origin. Thus, they have a lifelong highly elevated level of circulating low density lipoprotein (LDL) cholesterol which drives premature atherosclerosis. The high levels of LDL particles, often associated with an elevated level of circulating lipoprotein(a) particles, are both capable of inducing and maintaining endothelial dysfunction. Such pre-existing endothelial dysfunction can be exacerbated by exposure to SARS-CoV-2 viral particles, by exposure to fine particulate matter generated by climate change-associated wildfires, and by dehydration during deadly heatwaves linked to the globally rising temperatures. The external factors can severely worsen the pre-existing endothelial dysfunction, and thereby significantly increase the risk of a cardiovascular event in the exposed FH patients.
Assuntos
Aterosclerose , COVID-19 , Hipercolesterolemia , LDL-Colesterol , Endotélio , Humanos , Hipercolesterolemia/complicações , SARS-CoV-2RESUMO
The field of forensic DNA analysis has experienced significant advancements over the years, such as the advent of DNA fingerprinting, the introduction of the polymerase chain reaction for increased sensitivity, the shift to a primary genetic marker system based on short tandem repeats, and implementation of national DNA databases. Now, the forensics field is poised for another revolution with the advent of dense single nucleotide polymorphisms (SNPs) testing. SNP testing holds the potential to significantly enhance source attribution in forensic cases, particularly those involving low-quantity or low-quality samples. When coupled with genetic genealogy and kinship analysis, it can resolve countless active cases as well as cold cases and cases of unidentified human remains, which are hindered by the limitations of existing forensic capabilities that fail to generate viable investigative leads with DNA. The field of forensic genetic genealogy combined with genome-wide sequencing can associate relatives as distant as the seventh-degree and beyond. By leveraging volunteer-populated databases to locate near and distant relatives, genetic genealogy can effectively narrow the candidates linked to crime scene evidence or aid in determining the identity of human remains. With decreasing DNA sequencing costs and improving sensitivity of detection, forensic genetic genealogy is expanding its capabilities to generate investigative leads from a wide range of biological evidence.
Assuntos
Impressões Digitais de DNA , Genética Forense , Polimorfismo de Nucleotídeo Único , Humanos , Impressões Digitais de DNA/métodos , Genética Forense/métodos , LinhagemRESUMO
Over the past 30 years, forensic experts from Croatia and Bosnia and Herzegovina have embraced advanced technologies and innovations to enable great efficacy and proficiency in the identification of war victims. The wartime events in the countries of former Yugoslavia greatly influenced the application of the selected DNA analyses as routine tools for the identification of skeletal remains, especially those from mass graves. Initially, the work was challenging because of the magnitude of the events, technical aspects, and political aspects. Collaboration with reputable foreign forensic experts helped tremendously in the efforts to start applying DNA analysis routinely and with increasing success. In this article, we reviewed the most significant achievements related to the application of DNA analysis in identifying skeletal remains in situations where standard identification methods were insufficient.
Assuntos
Restos Mortais , Bósnia e Herzegóvina , Humanos , Croácia , Antropologia Forense/métodos , Guerra , Impressões Digitais de DNARESUMO
MOTIVATION: Read-merging algorithms that look solely at the reads can misalign and mis-merge the reads (especially near repetitive sequences). RESULTS: The C++ program ProSynAR has been written to take the reads' position in the reference into account when performing (and deciding whether to perform) a merge. AVAILABILITY: *Nix users can retrieve the source from GitHub (https://github.com/Benjamin-Crysup/prosynar). Windows binary available at https://github.com/Benjamin-Crysup/prosynar/releases/download/1.0/prosynar.zip. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Software , Análise de Sequência de DNA , Algoritmos , Sequências Repetitivas de Ácido NucleicoRESUMO
Y chromosome Short Tandem Repeat (STR) haplotypes have been used in assisting forensic investigations primarily for identification and male lineage determination. The current SWGDAM interpretation guidelines for Y-STR typing provide helpful guidance on those purposes but do not address the issue of kinship analysis with Y-STR haplotypes. Because of the high mutation rate of Y-STRs, there are complex missing person cases in which inconsistent Y-STR haplotypes between true paternal lineage relatives will arise and cases with two or more male references in the same lineage and yet differ in their haplotypes. Therefore, more useful methods are needed for interpreting the Y-STR haplotype data. Computational methods and interpretation guidelines have been developed specifically addressing this issue, either using a mismatch-based counting method or a pedigree likelihood ratio method. In this study, a software program, MPKin-YSTR, was developed by implementing those more sophisticated methods. This software should be able to improve the interpretation of complex cases with Y-STR haplotype evidence. Thus, more biological evidence will be interpreted, which in turn will result in more investigation leads to help solve crimes.
Assuntos
Cromossomos Humanos Y , Repetições de Microssatélites , Humanos , Masculino , Haplótipos/genética , Cromossomos Humanos Y/genética , Repetições de Microssatélites/genética , Linhagem , Genética PopulacionalRESUMO
A novel approach is presented that increases sensitivity and specificity for detecting minimal traces of DNA in liquid and on solid samples. Förster Resonance Energy Transfer (FRET) from YOYO to Ethidium Bromide (EtBr) substantially increases the signal from DNA-bound EtBr highly enhancing sensitivity and specificity for DNA detection. The long fluorescence lifetime of the EtBr acceptor, when bound to DNA, allows for multi-pulse pumping with time gated (MPPTG) detection, which highly increases the detectable signal of DNA-bound EtBr. A straightforward spectra/image subtraction eliminates sample background and allows for a huge increase in the overall detection sensitivity. Using a combination of FRET and MPPTG detection an amount as small as 10 pg of DNA in a microliter sample can be detected without any additional sample purification/manipulation or use of amplification technologies. This amount of DNA is comparable to the DNA content of a one to two human cells. Such a detection method based on simple optics opens the potential for robust, highly sensitive DNA detection/imaging in the field, quick evaluation/sorting (i.e., triaging) of collected DNA samples, and can support various diagnostic assays.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Substâncias Intercalantes , Humanos , Transferência Ressonante de Energia de Fluorescência/métodos , DNA , Sensibilidade e EspecificidadeRESUMO
Informed consent is based on basic ethical principles that should be considered when conducting biomedical and behavioral research involving human subjects. These principles-respect, beneficence, and justice-form the foundations of informed consent which in itself is grounded on three fundamental elements: information, comprehension, and voluntary participation. While informed consent has focused on human subjects and research, the practice has been adopted willingly in the forensic science arena primarily to acquire reference samples from family members to assist in identifying missing persons. With advances in molecular biology technologies, data mining, and access to metadata, it is important to assess whether the past informed consent process and in particular associated risks are concomitant with these increased capabilities. Given the state-of-the-art, areas in which informed consent may need to be modified and augmented are as follows: reference samples from family members in missing persons or unidentified human remains cases; targeted analysis of an individual(s) during forensic genetic genealogy cases to reduce an investigative burden; donors who provide their samples for validation studies (to include population studies and entry into databases that would be applied to forensic statistical calculations) to support implementation of procedures and operations of the forensic laboratory; family members that may contribute samples or obtain genetic information from a molecular autopsy; and use of medical and other acquired samples that could be informative for identification purposes. The informed consent process should cover (1) purpose for collection of samples; (2) process to analyze the samples (to include type of data); (3) benefits (to donor, target, family, community, etc. as applicable); (4) risks (to donor, target, family, community, etc. as applicable); (5) access to data/reports by the donor; (6) sample disposition; (7) removal of data process (i.e., expungement); (8) process to ask questions/assessment of comprehension; (9) follow-up processes; and (10) voluntary, signed, and dated consent. Issues surrounding these topics are discussed with an emphasis on addressing risk factors. Addressing informed consent will allow human subjects to make decisions voluntarily and with autonomy as well as secure the use of samples for intended use.
Assuntos
Compreensão , Consentimento Livre e Esclarecido , Humanos , Projetos de PesquisaRESUMO
Next-generation sequencing (NGS), also known as massively sequencing, enables large dense SNP panel analyses which generate the genetic component of forensic investigative genetic genealogy (FIGG). While the costs of implementing large SNP panel analyses into the laboratory system may seem high and daunting, the benefits of the technology may more than justify the investment. To determine if an infrastructural investment in public laboratories and using large SNP panel analyses would reap substantial benefits to society, a cost-benefit analysis (CBA) was performed. This CBA applied the logic that an increase of DNA profile uploads to a DNA database due to a sheer increase in number of markers and a greater sensitivity of detection afforded with NGS and a higher hit/association rate due to large SNP/kinship resolution and genealogy will increase investigative leads, will be more effective for identifying recidivists which in turn reduces future victims of crime, and will bring greater safety and security to communities. Analyses were performed for worst case/best case scenarios as well as by simulation sampling the range spaces with multiple input values simultaneously to generate best estimate summary statistics. This study shows that the benefits, both tangible and intangible, over the lifetime of an advanced database system would be huge and can be projected to be for less than $1 billion per year (over a 10-year period) investment can reap on average > $4.8 billion in tangible and intangible cost-benefits per year. More importantly, on average > 50,000 individuals need not become victims if FIGG were employed, assuming investigative associations generated were acted upon. The benefit to society is immense making the laboratory investment a nominal cost. The benefits likely are underestimated herein. There is latitude in the estimated costs, and even if they were doubled or tripled, there would still be substantial benefits gained with a FIGG-based approach. While the data used in this CBA are US centric (primarily because data were readily accessible), the model is generalizable and could be used by other jurisdictions to perform relevant and representative CBAs.
Assuntos
Impressões Digitais de DNA , DNA , Humanos , Análise Custo-Benefício , DNA/análise , Sequenciamento de Nucleotídeos em Larga Escala , Crime , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Tandem repeats (TR), highly variable genomic variants, are widely used in individual identification, disease diagnostics, and evolutionary studies. The recent advances in sequencing technologies and bioinformatic tools facilitate calling TR haplotypes genome widely. Both length-based and sequence-based TR alleles are used in different applications. However, sequence-based TR alleles could provide the highest precision in characterizing TR haplotypes. The need to identify the differences at the single nucleotide level between or among TR haplotypes with an easy-use bioinformatic tool is essential. RESULTS: In this study, we developed a Universal STR Allele Toolkit (USAT) for TR haplotype analysis, which takes TR haplotype output from existing tools to perform allele size conversion, sequence comparison of haplotypes, figure plotting, comparison for allele distribution, and interactive visualization. An exemplary application of USAT for analysis of the CODIS core STR loci for DNA forensics with benchmarking human individuals demonstrated the capabilities of USAT. USAT has user-friendly graphic interfaces and runs fast in major computing operating systems with parallel computing enabled. CONCLUSION: USAT is a user-friendly bioinformatics software for interpretation, visualization, and comparisons of TRs.
Assuntos
Biologia Computacional , Repetições de Microssatélites , Humanos , Alelos , Haplótipos , Análise de Sequência de DNARESUMO
This article presents a novel approach to increase the detection sensitivity of trace amounts of DNA in a sample by employing Förster resonance energy transfer (FRET) between intercalating dyes. Two intercalators that present efficient FRET were used to enhance sensitivity and improve specificity in detecting minute amounts of DNA. Comparison of steady-state acceptor emission spectra with and without the donor allows for simple and specific detection of DNA (acceptor bound to DNA) down to 100 pg/µL. When utilizing as an acceptor a dye with a significantly longer lifetime (e.g., ethidium bromide bound to DNA), multipulse pumping and time-gated detection enable imaging/visualization of picograms of DNA present in a microliter of an unprocessed sample or DNA collected on a swab or other substrate materials.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Substâncias Intercalantes , Corantes , DNA/genética , Etídio , Corantes FluorescentesRESUMO
MOTIVATION: Current read-mapping software uses a singular specification of alignment parameters with respect to the reference. In the presence of varying reference structures (such as the repetitive regions of the human genome), alignments can be improved if those parameters are allowed vary. RESULTS: To that end, the C++ program ProDerAl was written to refine previously generated alignments using varying parameters for these problematic regions. Synthetic benchmarks show that this realignment can result in an order of magnitude fewer misaligned bases. AVAILABILITY AND IMPLEMENTATION: *Nix users can retrieve the source from GitHub (https://github.com/Benjamin-Crysup/proderal.git). Windows binary available at https://github.com/Benjamin-Crysup/proderal/releases/download/v1.1/proderal.zip. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
RESUMO
The skin microbiome is a highly abundant and relatively stable source of DNA that may be utilized for human identification (HID). In this study, a set of single nucleotide polymorphisms (SNPs) with a high mean estimated Wright's fixation index (FST) (>0.1) and widespread abundance (found in ≥75% of samples compared) were selected from a diverse set of markers in the hidSkinPlex panel. The least absolute shrinkage and selection operator (LASSO) was used in a novel machine learning framework to generate a SNP panel and predict the human host from skin microbiome samples collected from the hand, manubrium, and foot. The framework was devised to emulate a new unknown person introduced to the algorithm and to match samples from that person against a population database. Unknown samples were classified with 96% accuracy (Matthews correlation coefficient [MCC], 0.954) in the test (n = 225 samples) data set. A final panel of informative SNPs was determined for HID (hidSkinPlex+) using all 51 individuals sampled at three body sites in triplicate. The hidSkinPlex+ panel comprises 365 SNPs and yielded prediction accuracy for the correct host of 95% (MCC = 0.949). The accuracy of the hidSkinPlex+ panel may be somewhat overestimated due to using 26 individuals from the training data set for the selection of the final panel. However, this accuracy still provides an indication of performance when tested on new samples. IMPORTANCE One of the fundamental goals in forensic genetics is to identify the source of biological evidence. Methods for detecting human DNA have advanced and can be quite sensitive, but not all DNA samples are amenable to current methods. However, the human skin microbiome is a source of DNA with high copy numbers, and it has the potential for high discriminatory power. The hidSkinPlex panel has been used for HID; however, some aspects of it could be improved. Missing information is ambiguous, as it is unclear if marker drop-out is a by-product of a low-template sample or if the reasons for not observing a marker are biological. Such ambiguity may confound methods for HID, and as such, an improved marker set (hidSkinPlex+) was designed that is considerably smaller and more robust to drop-out (365 SNPs contained in 135 markers) yet still can be used to accurately predict the human host.
Assuntos
Microbiota , Polimorfismo de Nucleotídeo Único , DNA , Antropologia Forense , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Microbiota/genética , Análise de Sequência de DNARESUMO
Laboratories and their criminal justice systems are confronted with challenges for implementing new technologies, practices, and policies even when there appears to be demonstrative benefits to operational performance. Impacting decisions are the often higher costs associated with, for example, new technologies, limited current budgets, and making hard decisions on what to sacrifice to take on the seemingly better approach. A prospective cost-benefit analysis (CBA) could help an agency better formulate its strategies and plans and more importantly delineate how a relatively small increase to take on, for example, a new technology can have large impact on the system (e.g., the agency, other agencies, victims and families, and taxpayers). To demonstrate the process and potential value a CBA was performed on the use of an alternate and more expensive swab with reported better DNA yield and being certified human DNA free (i.e., nylon 4N6FLOQSwabs®), versus the traditional less costly swab (i.e., cotton swab). Assumptions are described, potential underestimates and overestimates noted, different values applied (for low and modest to high), and potential benefits (monetary and qualitative) presented. The overall outcome is that the cost of using the more expensive technology pales compared with the potential tangible and intangible benefits. This approach could be a guide for laboratories (and associated criminal justice systems) worldwide to support increased funding, although the costs and benefits may vary locally and for different technologies, practices, and policies. With well-developed CBAs, goals of providing the best services to support the criminal justice system and society can be attained.
Assuntos
Nylons , Análise Custo-Benefício , Humanos , Estudos ProspectivosRESUMO
Rapid DNA platforms are fully automated systems capable of processing DNA from biological samples and interpreting the results in approximately 90 minutes with minimal human intervention. With a greater reliance on the system than on the analyst, validation data are especially needed to define the performance and limitations of commercially available Rapid DNA systems. Thus, validation studies of a Rapid DNA workflow consisting of the Applied Biosystems RapidHIT ID Instrument and RapidLINK software with a focus on the ACE GlobalFiler Express Sample Cartridge and reference buccal swabs were performed in accordance with Scientific Working Group on DNA Analysis Methods Validation Guidelines. These validation studies included assessments of sensitivity, contamination, concordance, reproducibility and repeatability, stability, inhibition, mixtures, sample reprocessing, precision, and first-pass success rate. Overall, the current Applied Biosystems RapidHIT ID Instrument with the ACE GlobalFiler Express sample cartridge was found to be a reliable tool for generation of STR profiles from reference-type buccal swabs.
Assuntos
Impressões Digitais de DNA , Repetições de Microssatélites , DNA/genética , Impressões Digitais de DNA/métodos , Humanos , Reprodutibilidade dos Testes , SoftwareRESUMO
With the advent of expanded STR (short tandem repeats) typing kits, it was necessary to determine allele frequencies and other appropriate population data parameters for El Salvador. Samples were collected from the central, east, and west regions of the country and typed for 21 forensically relevant STR loci. The data indicate that all loci are highly polymorphic, the three regions are genetically similar, and the population data are similar to those of US Hispanics. The results of this study support that the allele frequency data described herein can be used for statistical calculations for human identity testing in El Salvador.
Assuntos
Impressões Digitais de DNA , Genética Populacional , Frequência do Gene , Hispânico ou Latino , Humanos , Repetições de MicrossatélitesRESUMO
MOTIVATION: Assays in mitochondrial genomics rely on accurate read mapping and variant calling. However, there are known and unknown nuclear paralogs that have fundamentally different genetic properties than that of the mitochondrial genome. Such paralogs complicate the interpretation of mitochondrial genome data and confound variant calling. RESULTS: Remove the Numts! (RtN!) was developed to categorize reads from massively parallel sequencing data not based on the expected properties and sequence identities of paralogous nuclear encoded mitochondrial sequences, but instead using sequence similarity to a large database of publicly available mitochondrial genomes. RtN! removes low-level sequencing noise and mitochondrial paralogs while not impacting variant calling, while competing methods were shown to remove true variants from mitochondrial mixtures. AVAILABILITY AND IMPLEMENTATION: https://github.com/Ahhgust/RtN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Genoma Mitocondrial , Sequenciamento de Nucleotídeos em Larga Escala , Núcleo Celular , Análise de Sequência de DNA , SoftwareRESUMO
Microbial DNA, shed from human skin, can be distinctive to its host and, thus, help individualize donors of forensic biological evidence. Previous studies have utilized single-locus microbial DNA markers (e.g., 16S rRNA) to assess the presence/absence of personal microbiota to profile human hosts. However, since the taxonomic composition of the microbiome is in constant fluctuation, this approach may not be sufficiently robust for human identification (HID). Multimarker approaches may be more powerful. Additionally, genetic differentiation, rather than taxonomic distinction, may be more individualizing. To this end, the nondominant hands of 51 individuals were sampled in triplicate (n = 153). They were analyzed for markers in the hidSkinPlex, a multiplex panel comprising candidate markers for skin microbiome profiling. Single-nucleotide polymorphisms (SNPs) with the highest Wright's fixation index (FST) estimates were then selected for predicting donor identity using a support vector machine (SVM) learning model. FST is an estimate of the genetic differences within and between populations. Three different SNP selection criteria were employed: SNPs with the highest-ranking FST estimates (i) common between any two samples regardless of markers present (termed overall); (ii) each marker common between samples (termed per marker); and (iii) common to all samples used to train the SVM algorithm for HID (termed selected). The SNPs chosen based on criteria for overall, per marker, and selected methods resulted in an accuracy of 92.00%, 94.77%, and 88.00%, respectively. The results support that estimates of FST, combined with SVM, can notably improve forensic HID via skin microbiome profiling. IMPORTANCE There is a need for additional genetic information to help identify the source of biological evidence found at a crime scene. The human skin microbiome is a potentially abundant source of DNA that can enable the identification of a donor of biological evidence. With microbial profiling for human identification, there will be an additional source of DNA to identify individuals as well as to exclude individuals wrongly associated with biological evidence, thereby improving the utility of forensic DNA profiling to support criminal investigations.
Assuntos
Microbiota , Pele/microbiologia , Bactérias/genética , Antropologia Forense , Humanos , Aprendizado de Máquina , Polimorfismo de Nucleotídeo Único , Máquina de Vetores de SuporteRESUMO
Deconvoluting mixture samples is one of the most challenging problems confronting DNA forensic laboratories. Efforts have been made to provide solutions regarding mixture interpretation. The probabilistic interpretation of Short Tandem Repeat (STR) profiles has increased the number of complex mixtures that can be analyzed. A portion of complex mixture profiles, particularly for mixtures with a high number of contributors, are still being deemed uninterpretable. Novel forensic markers, such as Single Nucleotide Variants (SNV) and microhaplotypes, also have been proposed to allow for better mixture interpretation. However, these markers have both a lower discrimination power compared with STRs and are not compatible with CODIS or other national DNA databanks worldwide. The short-read sequencing (SRS) technologies can facilitate mixture interpretation by identifying intra-allelic variations within STRs. Unfortunately, the short size of the amplicons containing STR markers and sequence reads limit the alleles that can be attained per STR. The latest long-read sequencing (LRS) technologies can overcome this limitation in some samples in which larger DNA fragments (including both STRs and SNVs) with definitive phasing are available. Based on the LRS technologies, this study developed a novel CODIS compatible forensic marker, called a macrohaplotype, which combines a CODIS STR and flanking variants to offer extremely high number of haplotypes and hence very high discrimination power per marker. The macrohaplotype will substantially improve mixture interpretation capabilities. Based on publicly accessible data, a panel of 20 macrohaplotypes with sizes of ~ 8 k bp and the maximum high discrimination powers were designed. The statistical evaluation demonstrates that these macrohaplotypes substantially outperform CODIS STRs for mixture interpretation, particularly for mixtures with a high number of contributors, as well as other forensic applications. Based on these results, efforts should be undertaken to build a complete workflow, both wet-lab and bioinformatics, to precisely call the variants and generate the macrohaplotypes based on the LRS technologies.