Massive Parallel Sequencing Technologies Research Articles

Next-generation sequencing in Charcot-Marie-Tooth: a proposal for improvement of ACMG guidelines for variant evaluation

BackgroundThe application of massive parallel sequencing technologies in the molecular analysis of Charcot-Marie-Tooth (CMT) has enabled the rapid and cost-effective identification of numerous potentially significant variants for diagnostic purposes. The...

A comparison of likelihood ratios calculated from surface DNA mixtures using MPS and CE Technologies

This study evaluates the performance of analysing surface DNA samples using massively parallel sequencing (MPS) compared to traditional capillary electrophoresis (CE). A total of 30 samples were collected from various surfaces in an office environment and were analysed with CE and MPS. These were compared against 60 reference samples (office inhabitants). To identify contributors, likelihood ratios (LRs) were calculated for MPS and CE data using the probabilistic genotyping software MPSproto and EuroForMix respectively. Although a higher number of sequences/peaks were observed per DNA profile in MPS compared to CE, LR values were found to be lower for MPS data formats. This might be the result of the increased complexity of MPS data, along with a possible elevation of unknown alleles and/or artefacts. The study highlights avenues for improving MPS data quality and analysis to facilitate more robust interpretation of challenging casework-like samples.

Epithelioid Hemangioma of the Spine: A Case Series and Treatment Flow Chart-Experience from a Single Centre.

Epithelioid hemangioma is recognized by the World Health Organization as a distinct benign neoplasm; however, it is characterized by locally aggressive and rarely metastasizing behavior. Epithelioid vascular tumors are rare bony vascular lesions with varying degrees of malignant potential that remain controversial because of their rarity, unusual morphological features, and unpredictable biological behavior. The application of new molecular tools, such as massive parallel sequencing technologies, have provided new diagnostic markers and an opportunity to further refine the classification of bone vascular neoplasms. Very few cases of EH of the spine have been reported in the literature; therefore, it is difficult to make evidence-based therapeutic decisions for these patients. We report herein our experience with eleven patients suffering from EH of the spine. The study population included three males and eight females treated in our center from 2016 to the present; the average age was 44.8 years (range 14-75 years). The surgical, clinical, and radiographic data were retrospectively analyzed. The mean follow-up was 34.8 months. All patients presented lytic vertebral body lesions, six of them with pathological fracture. The majority of patients (80%) presented myelo-radicular compression. All patients were surgically treated, and preoperative embolization was performed in all cases. In light of the literature review and the clinical experience of our center, we can consider EH a locally aggressive tumor that requires surgical treatment in case of symptoms. Here, we propose a treatment algorithm that could be useful in the management of patients with this rare disease.

Population genetic analyses of Eastern Chinese Han nationality using ForenSeq™ DNA Signature Prep Kit.

Currently, the most commonly used method for human identification and kinship analysis in forensic genetics is the detection of length polymorphism in short tandem repeats (STRs) using polymerase chain reaction (PCR) and capillary electrophoresis (CE). However, numerous studies have shown that considerable sequence variations exist in the repeat and flanking regions of the STR loci, which cannot be identified by CE detection. Comparatively, massively parallel sequencing (MPS) technology can capture these sequence differences, thereby enhancing the identification capability of certain STRs. In this study, we used the ForenSeq™ DNA Signature Prep Kit to sequence 58 STRs and 94 individual identification SNPs (iiSNPs) in a sample of 220 unrelated individuals from the Eastern Chinese Han population. Our aim is to obtain MPS-based STR and SNP data, providing further evidence for the study of population genetics and forensic applications. The results showed that the MPS method, utilizing sequence information, identified a total of 486 alleles on autosomal STRs (A-STRs), 97 alleles on X-chromosome STRs (X-STRs), and 218 alleles on Y-chromosome STRs (Y-STRs). Compared with length polymorphism, we observed an increase of 260 alleles (157, 31, and 72 alleles on A-STRs, X-STRs, and Y-STRs, respectively) across 36 STRs. The most substantial increments were observed in DYF387S1 and DYS389II, with increases of 287.5% and 250%, respectively. The most increment in the number of alleles was found at DYF387S1 and DYS389II (287.5% and 250%, respectively). The length-based (LB) and sequence-based (SB) combined random match probability (RMP) of 27 A-STRs were 6.05E-31 and 1.53E-34, respectively. Furthermore, other forensic parameters such as total discrimination power (TDP), cumulative probability of exclusion of trios (CPEtrio), and duos (CPEduo) were significantly improved when using the SB data, and informative data were obtained for the 94 iiSNPs. Collectively, these findings highlight the advantages of MPS technology in forensic genetics, and the Eastern Chinese Han genetic data generated in this study could be used as a valuable reference for future research in this field.

Utilizing Massively Parallel Sequencing (MPS) of Human Leukocyte Antigen (HLA) Gene Polymorphism to Assess Relatedness in Deficiency Parentage Testing.

In the realm of DNA testing with legal implications, the reliability and precision of genetic markers play a pivotal role in confirming or negating paternity claims. This study aimed to assess the potential utility of human leukocyte antigen (HLA) gene polymorphism through massively parallel sequencing (MPS) technology as robust forensic markers for parentage testing involving genetic deficiencies. It sought to redefine the significance of HLA genes in this context. Data on autosomal short tandem repeat (aSTR) mutational events across 18 paternity cases involving 16 commonly employed microsatellite loci were presented. In instances where traditional aSTR analysis failed to establish statistical certainty, kinship determination was pursued via HLA genotyping, encompassing the amplification of 17 linked HLA loci. Within the framework of this investigation, phase-resolved genotypes for HLA genes were meticulously generated, resulting in the definition of 34 inherited HLA haplotypes. An impressive total of 274 unique HLA alleles, which were classified at either the field 3 or 4 level, were identified, including the discovery of four novel HLA alleles. Likelihood ratio (LR) values, which indicated the likelihood of the observed data under a true biological relationship versus no relationship, were subsequently calculated. The analysis of the LR values demonstrated that the HLA genes significantly enhanced kinship determination compared with the aSTR analysis. Combining LR values from aSTR markers and HLA loci yielded conclusive outcomes in duo paternity cases, showcasing the potential of HLA genes and MPS technology for deeper insights and diversity in genetic testing. Comprehensive reference databases and high-resolution HLA typing across diverse populations are essential. Reintegrating HLA alleles into forensic identification complements existing markers, creating a potent method for future forensic analysis.

A Multisample Approach in Forensic Phenotyping of Chronological Old Skeletal Remains Using Massive Parallel Sequencing (MPS) Technology.

It is very important to generate phenotypic results that are reliable when processing chronological old skeletal remains for cases involving the identification of missing persons. To improve the success of pigmentation prediction in Second World War victims, three bones from each of the eight skeletons analyzed were included in the study, which makes it possible to generate a consensus profile. The PowerQuant System was used for quantification, the ESI 17 Fast System was used for STR typing, and a customized version of the HIrisPlex panel was used for PCR-MPS. The HID Ion Chef Instrument was used for library preparation and templating. Sequencing was performed with the Ion GeneStudio S5 System. Identical full profiles and identical hair and eye color predictions were achieved from three bones analyzed per skeleton. Blue eye color was predicted in five skeletons and brown in three skeletons. Blond hair color was predicted in one skeleton, blond to dark blond in three skeletons, brown to dark brown in two skeletons, and dark brown to black in two skeletons. The reproducibility and reliability of the results proved the multisample analysis method to be beneficial for phenotyping chronological old skeletons because differences in DNA yields in different bone types provide a greater possibility of obtaining a better-quality consensus profile.

Identification of miRnas with possible prognostic roles for HAM/TSP

ABSTRACT Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropic spastic paraparesis (HAM/TSP) is an insidiously progressive spinal cord disease for which there is no effective treatment. There is great interest in developing potential biomarkers to predict the pathogenesis of HAM/TSP disease. In this study, Illumina Massive Parallel Sequencing (MPS) technology was used to investigate the cellular global noncoding RNAome expression profile in HAM/TSP patients (n = 10), asymptomatic HTLV-1-infected carriers (ASP, n = 8), and a second group of healthy controls (n = 5). Various bioinformatics tools were used to align, annotate, and profile the sRNA-MPS reads. Among the 402 sRNAs detected, 251 were known and 50 were potentially novel sRNAs in the HAM and ASP groups compared with the HC group. Sixty-eight known sRNAs were significantly different between the ASP and HAM groups. Eighty-eight mature miRNAs were downregulated in subjects from HAM compared with ASP. Three of these miRs (hsa-miR-185-5p, 32-5p, and 192-5p) have the potential to be used as biomarkers for predicting the pathogenesis of HAM/TSP. The seven most deregulated miRs target genes have been associated with a variety of biological processes and molecular functions. The reactome pathways relevant to our findings provide a rich source of data and offer the opportunity to better understand sRNA regulation and function in HTLV-1 pathophysiology. To the best of our knowledge, this study is the first to demonstrate evaluates sRNAs in HTLV-1 patients with HAM/TSP.

Eye and hair color prediction of an early medieval adult and subadult skeleton using massive parallel sequencing technology.

Phenotypic trait prediction in ancient DNA analysis can provide information about the external appearance of individuals from past human populations. Some studies predicting eye and hair color in ancient adult skeletons have been published, but not for ancient subadult skeletons, which are more prone to decay. In this study, eye and hair color were predicted for an early medieval adult skeleton and a subadult skeleton that was anthropologically characterized as a middle-aged man and a subadult of unknown sex about 6years old. When processing the petrous bones, precautions were taken to prevent contamination with modern DNA. The MillMix tissue homogenizer was used for grinding, 0.5g of bone powder was decalcified, and DNA was purified in Biorobot EZ1. The PowerQuant System was used for quantification and a customized version of the HIrisPlex panel for massive parallel sequencing (MPS) analysis. Library preparation and templating were performed on the HID Ion Chef Instrument and sequencing on the Ion GeneStudio S5 System. Up to 21ng DNA/g of powder was obtained from ancient petrous bones. Clean negative controls and no matches with elimination database profiles confirmed no contamination issue. Brown eyes and dark brown or black hair were predicted for the adult skeleton and blue eyes and brown or dark brown hair for the subadult skeleton. The MPS analysis results obtained proved that it is possible to predict hair and eye color not only for an adult from the Early Middle Ages, but also for a subadult skeleton dating to this period.

Test development, optimization and validation of a WGS pipeline for genetic disorders

BackgroundWith advances in massive parallel sequencing (MPS) technology, whole-genome sequencing (WGS) has gradually evolved into the first-tier diagnostic test for genetic disorders. However, deployment practice and pipeline testing for clinical WGS are lacking.MethodsIn this study, we introduced a whole WGS pipeline for genetic disorders, which included the entire process from obtaining a sample to clinical reporting. All samples that underwent WGS were constructed using polymerase chain reaction (PCR)-free library preparation protocols and sequenced on the MGISEQ-2000 platform. Bioinformatics pipelines were developed for the simultaneous detection of various types of variants, including single nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs) and balanced rearrangements, mitochondrial (MT) variants, and other complex variants such as repeat expansion, pseudogenes and absence of heterozygosity (AOH). A semiautomatic pipeline was developed for the interpretation of potential SNVs and CNVs. Forty-five samples (including 14 positive commercially available samples, 23 laboratory-held positive cell lines and 8 clinical cases) with known variants were used to validate the whole pipeline.ResultsIn this study, a whole WGS pipeline for genetic disorders was developed and optimized. Forty-five samples with known variants (6 with SNVs and Indels, 3 with MT variants, 5 with aneuploidies, 1 with triploidy, 23 with CNVs, 5 with balanced rearrangements, 2 with repeat expansions, 1 with AOHs, and 1 with exon 7–8 deletion of SMN1 gene) validated the effectiveness of our pipeline.ConclusionsThis study has been piloted in test development, optimization, and validation of the WGS pipeline for genetic disorders. A set of best practices were recommended using our pipeline, along with a dataset of positive samples for benchmarking.

Opportunities and achievements of using massive parallel sequencing in the diagnosis of neurodevelopmental diseases

The contribution of genetic factors to development of neurological diseases has long been recognized, and the majority of the advances coupled to using molecular genetic technologies resulted in identifying genes associated with the pathology of developing nervous system. The review is aimed at demonstrating opportunities and achievements of using massive parallel sequencing technology in the diagnosis of a disease with damage to nervous system. Currently, next-generation sequencing (NGS) technology in the genetic diagnosis of epilepsy relies on targeted gene panels, whole exome sequencing or whole genome sequencing. Integrating genetic and genomic analysis into clinical practice to establish clear molecular diagnoses for previously undiagnosed patients will avoid further unnecessary diagnosis and therefore lead to greater healthcare cost savings over time. The use of biomarkers will contribute to the prediction of disease outcome and therapy decision-making.

The utility of dust for forensic intelligence: Exploring collection methods and detection limits for environmental DNA, elemental and mineralogical analyses of dust samples

Environmental DNA (eDNA), elemental and mineralogical analyses of soil have been shown to be specific to their source material, prompting consideration of using the airborne fraction of soil (dust) for forensic intelligence work. Dust is ubiquitous in the environment and is easily transferred to items belonging to a person of interest, making dust analysis an ideal tool in forensic casework. The advent of Massive Parallel Sequencing technologies means metabarcoding of eDNA can uncover bacterial, fungal, and even plant genetic fingerprints in dust particles. Combining this with elemental and mineralogical compositions offers multiple, complementary lines of evidence for tracing the origin of an unknown dust sample. This is particularly pertinent when recovering dust from a person of interest to ascertain where they may have travelled. Prior to proposing dust as a forensic trace material, however, the optimum sampling protocols and detection limits need to be established to place parameters around its utility in this context. We tested several approaches to collecting dust from different materials and determined the lowest quantity of dust that could be analysed for eDNA, elemental composition and mineralogy, whilst still yielding results capable of distinguishing between sites. We found that fungal eDNA profiles could be obtained from multiple sample types and that tape lifts were the optimum collection method for discriminating between sites. We successfully recovered both fungal and bacterial eDNA profiles down to 3 mg of dust (the lowest tested quantity) and recovered elemental and mineralogical compositions for all tested sample quantities. We show that dust can be reliably recovered from different sample types, using different sampling techniques, and that fungi and bacteria, as well as elemental and mineralogical profiles, can be generated from small sample quantities, highlighting the utility of dust for forensic intelligence.

Gerstmann–Sträussler–Scheinker syndrome with early-onset spinocerebellar ataxia phenotype

The article presents a description of the family case of rare hereditary prion disease – Gerstmann–Sträussler–Scheinker syndrome with a verified p.P102L mutation in the PRNP gene. The clinical picture was represented by progressive cerebellar ataxia, pyramidal signs, bulbar syndrome, and neuropathy, which made it possible to establish a preliminary diagnosis of autosomal dominant spinocerebellar ataxia. Subsequently, the final diagnosis was verified using the massive parallel sequencing technology. The features of this observation are the variable age of onset within the family, with the unusually early age of the disease onset in the proband (age of 25 years), the absence of cognitive impairment, as well as the absence of pathological changes in the electroencephalography and brain MRI study. The possible modifying role of polymorphism in codon 129 of the PRNP gene (129Val) in the disease phenotype formation, as well as the clinical findings and diagnostic methods of this syndrome are discussed.

Application Progress of Massively Parallel Sequencing Technology in STR Genetic Marker Detection.

In recent years, more and more forensic genetics laboratories have begun to apply massively parallel sequencing (MPS) technology, that is, next-generation sequencing (NGS) technology, to detect common forensic genetic markers, including short tandem repeat (STR), single nucleotide polymorphism (SNP), the control region or whole genome of mitochondrial DNA (mtDNA), as well as messenger RNA (mRNA), etc., for forensic practice, such as individual identification, kinship analysis, ancestry inference and body fluid identification. As the most widely used genetic marker in forensic genetics, STR is currently mainly detected by capillary electrophoresis (CE) platform. Compared with CE platform, MPS technology has the advantages of simultaneous detection of a large number of genetic markers, massively parallel detection of samples, the polymorphism of sequence detected by NGS makes STR have the advantages of higher resolution and system efficiency. However, MPS technology is expensive, there is no uniform standard so far, and there are problems such as how to integrate MPS-STR data with the existing CE-STR database. This review summarizes the current status of the application of MPS technology in the detection of STR genetic markers in forensic genetics, puts forward the main problems that need to be solved urgently, and prospects the application prospect of this technology in forensic genetics.

Microhaplotype and Y-SNP/STR (MY): A novel MPS-based system for genotype pattern recognition in two-person DNA mixtures

BackgroundsY-chromosomal haplotypes based on Y-short tandem repeats (STRs) and Y-single nucleotide polymorphisms/insertion and deletion polymorphisms (SNPs/InDels) are used to characterize paternal lineages of unknown male trace donors. However, Y-chromosomal genetic markers are not currently sufficient for precise individual identification. Microhaplotype (MH), generally < 200 bp on autosomes and consisting of two or more SNPs, was recently introduced in forensic genetics with the development of massive parallel sequencing technology and may facilitate identification and DNA mixture deconvolution. Therefore, combining the two kinds of genetic markers may be beneficial in many forensic scenarios, especially crime scenes with male suspects, such as sexual assault cases. MethodsIn the present study, we developed a novel MPS-based panel, Microhaplotype and Y-SNP/STR (MY), by multiplex PCR and 150-bp paired-end sequencing, including 114 Y-SNPs (twelve dominant Y-DNA haplogroups), 45 Y-STRs (N-1 stutter < 0.09; estimated mutation rate < 5 × 10−3), and 22 MHs (allele coverage ratio > 0.91; pairwise distance > 10 Mb). Additionally, MY system-based genotype pattern recognition (GPR), a regression-based method to identify the genotype pattern for each MH locus, is proposed for two-person DNA mixture deconvolution. We integrated 26 two-person genotype combinations into nine genotype patterns and validated the application range of GPR based on DNA profiles of ten sets of simulated male-male DNA mixtures (1:10–1:2). ResultsThe effective number of alleles (Ae) ranged from 3.62 to 14.72, with an average of 7.17, in 100 Chinese Guangdong Han individuals. The cumulative discrimination power was 1–5.00 × 10−31, and the cumulative power of exclusion was 1–5.00 × 10−8 and 1–4.85 × 10−12 for duo and trio paternity testing, respectively. Furthermore, the actual mixing ratio-depth of coverage (DoC) ratio (RDoC) regression relationships were established for different genetic markers and genotype patterns. In five overlapping areas, genotype differentiation of the major and minor contributors required likelihood ratio methods. In nonoverlapping areas, the genotype pattern could be recognized by comparing the observed RDoC and RDoC ranges. ConclusionThe GPR can be used to deconvolute two-person DNA mixtures (application range: 1:10–1:2) for individual identification.

National Genome Initiatives in Europe and the United Kingdom in the Era of Whole-Genome Sequencing: A Comprehensive Review.

Identification of genomic variability in population plays an important role in the clinical diagnostics of human genetic diseases. Thanks to rapid technological development in the field of massive parallel sequencing technologies, also known as next-generation sequencing (NGS), complex genomic analyses are now easier and cheaper than ever before, which consequently leads to more effective utilization of these techniques in clinical practice. However, interpretation of data from NGS is still challenging due to several issues caused by natural variability of DNA sequences in human populations. Therefore, development and realization of projects focused on description of genetic variability of local population (often called “national or digital genome”) with a NGS technique is one of the best approaches to address this problem. The next step of the process is to share such data via publicly available databases. Such databases are important for the interpretation of variants with unknown significance or (likely) pathogenic variants in rare diseases or cancer or generally for identification of pathological variants in a patient’s genome. In this paper, we have compiled an overview of published results of local genome sequencing projects from United Kingdom and Europe together with future plans and perspectives for newly announced ones.

Clinical exome sequencing-Mistakes and caveats.

Massive parallel sequencing technology has become the predominant technique for genetic diagnostics and research. Many genetic laboratories have wrestled with the challenges of setting up genetic testing workflows based on a completely new technology. The learning curve we went through as a laboratory was accompanied by growing pains while we gained new knowledge and expertise. Here we discuss some important mistakes that have been made in our laboratory through 10 years of clinical exome sequencing but that have given us important new insights on how to adapt our working methods. We provide these examples and the lessons that we learned to help other laboratories avoid to make the same mistakes.

RNAseq of Infected Erythrocyte Surface Antigen-Encoding Genes.

Massive parallel sequencing technology has greatly increased the breadth and depth of transcriptomic data that can be captured from P. falciparum samples. This has revolutionized in vitro studies but uptake has been slower in the analysis of clinical samples. The principal barriers are the removal of contaminating white blood cells in a malaria endemic setting and preservation of the RNA. We provide here detailed methods for the collection of purified infected erythrocytes and the preservation and extraction of RNA. We also provide methods for assessing and addressing contaminating RNA from erythroid cells, and a protocol for RNAseq library preparation optimized to maximize yield from low amounts of parasite mRNA. Finally, we provide some examples of RNAseq library characteristics that may fail quality control for other species but are in fact satisfactory for P. falciparum RNAseq.

IDH1/2 Mutations in Patients With Diffuse Gliomas: A Single Centre Retrospective Massively Parallel Sequencing Analysis.

Patients below 55 years were genetically studied because the prevalence of isocitrate dehydrogenase 1 (IDH1) decreases in older patients and on grounds of cost-effectiveness, as suggested by the World Health Organization (WHO) in 2016. The aim of our study was to use novel massively parallel sequencing (MPS) approaches to examine rare variants of IDH1/2 in Czech diffuse astrocytic and oligodendroglial tumors (gliomas) patients below 55 years of age who had been immunohistochemically (IHC) diagnosed as IDH1 R132H negative. The IHC IDH1 status (wild type or mutant) of 275 tissue samples was analyzed using antibodies against the IDH1 R132H protein. Sixty-three samples of 55 years old patients with IHC IDH1 WT status were genotyped using two different MPS technologies to detect rare IDH1 and IDH2 variants. The tiered IHC (60 positive) and molecular (10 positive) approach thus revealed that 70 of the 275 samples (25%) bore IDH1/IDH2 mutations. The combined molecular and IHC approach thus revealed that 70 of the 275 samples (25%) considered in the study bore IDH1/IDH2 mutations. IHC detection of the IDH1 R132H variant should be routinely complemented with MPS to detect rare IDH1/2 variants in glioma patients below 55 years of age with negative IHC result of IDH R132H variant.

Application of Massive Parallel Sequencing Technology in Forensics: Comparative Analysis of Sequencing Platforms

Application of Massive Parallel Sequencing Technology in Forensics: Comparative Analysis of Sequencing Platforms

Development and validation of a novel 133-plex forensic STR panel (52 STRs and 81 Y-STRs) using single-end 400bp massive parallel sequencing.

Short tandem repeats (STRs) are the preferred genetic markers in forensic DNA analysis, routinely measured by capillary electrophoresis (CE) method based on the fragment length features. While, the massive parallel sequencing (MPS) technology could simultaneously target a large number of intriguing forensic STRs, bypassing the intrinsic limitations of amplicon size separation and accessible fluorophores in CE, which is efficient and promising for enabling the identification of forensic biological evidence. Here, we developed a novel MPS-based Forensic Analysis System Multiplecues SetB Kit of 133-plex forensic STR markers (52 STRs and 81 Y-STRs) and one Y-InDel (M175) based on multiplex PCR and single-end 400bp sequencing strategy. This panel was subjected to developmental validation studies according to the SWGDAM Validation Guidelines. Approximately 2185 MPS-based reactions using 6 human DNA standards and 8 male donors were conducted for substrate studies (filter paper, gauze, cotton swab, four different types of FTA cards, peripheral venous blood, saliva, and exfoliated cells), sensitivity studies (from 2ng down to 0.0625ng), mixture studies (two-person DNA mixtures), PCR inhibitor studies (seven commonly encountered PCR inhibitors), species specificity studies (11 non-human species), and repeatability studies. Results of concordance studies (413 Han males and 6 human DNA standards) generated by STRait Razor and in-house Python scripts indicated 99.98% concordance rate in STR calling relative to CE for STRs between 41,900 genotypes at 100 STR markers. Moreover, the limitations of present studies, the nomenclature rules and forensic MPS applications were also described. In conclusion, the validation studies based on ~ 2200 MPS-based and ~ 2500 CE-based DNA profiles demonstrated that the novel MPS-based panel meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities, and the STR nomenclature rules should be further regulated to integrate the inconformity between MPS-based and CE-based methods.