Next-generation Sequencing Platforms Research Articles

Economic implications of ddPCR and NGS-based noninvasive prenatal testing for fetal aneuploidy screening

Noninvasive prenatal testing (NIPT) heralds a transformative era in prenatal care, revolutionizing fetal health assessment. The recent adoption of aneuploidy screening signifies a significant advancement in prenatal genetic care in India. The current study aimed to assess the cost-effectiveness of ddPCR-based NIPT for detecting chromosome aneuploidies, comparing it with the next-generation sequencing (NGS) platform. This study adopts a laboratory-based observational approach to investigate the cost implications of NIPT for trisomies 13, 18, and 21 using ddPCR and NGS technologies. A meticulously designed cost analysis methodology was employed, adhering to established standards. The yearly capital and operational costs of NIPT were calculated with precision, focusing on the specific methods associated with ddPCR and NGS. The calculated annual capital and operating costs for NIPT using the ddPCR were $16,411 and $246,540 while those using the NGS platform were $91,440 and $250,560, respectively. The total cost of NIPT using ddPCR for 2,400 tests was $262,951, with an estimated cost per test of $110. In contrast, the total cost of NIPT using the NGS platform for 600 tests was $342,000, resulting in an estimated cost per test of $570. The ddPCR is five times more cost-effective. Moreover, it exhibits a fourfold reduction in time expenditure, attributable to streamlined procedures and does not require a complex bioinformatics analysis compared to the NGS. Moreover, this preliminary outcome on cost analysis for NIPT using ddPCR, as opposed to the NGS platform, can be extended to the health technology assessment (HTA) perspective for prenatal screening programs.

Parental Reconstruction from a Half-Sib Population of Stoneless Jujube Ziziphus jujuba Mill. Based on Individual Specific SNP Markers Using Multiplex PCR

The selection of unique and individual-specific SNPs is important as compared with universal SNPs for individual identification. Therefore, the main significance of this research is the selection of specific SNPs in male parent and the identification of offspring with these specific SNPs in their genome by multiplex PCR, which is utilized for genotyping of 332 half-sib plants of Ziziphus jujuba.This cost-effective method makes as much as possible to utilize the same amount of each pair of various targeted loci primers. After PCR amplification of targeted genome parts, the mixed products can be directly used in a next-generation sequencing platform. We concomitantly amplified 10 unique SNP loci at 10 different chromosomes of male JingZao 39 plants in 332 half-sib plants and sequenced them on the Illumina Novaseq 6000 platform. Analysis of SNP genotyping accuracy of 332 half-sib plants showed that all 10 unique SNPs in all 332 plants were correctly amplified in this multiplex PCR method. Furthermore, based on Mendelian inheritance, we identified 124 full-sib plants that have 10 unique SNPs in their genomes. These results were further confirmed by whole genome resequencing of 82 randomly selected half-sib plants, and the identity-by-descent values of all full-sib plants were between 0.4399 to 0.5652. This study displayed a cost-effective multiplex PCR method and proper identification of pollen parent through specific SNPs in half-sib progenies and firstly obtained a full-sib population between ‘Wuhezao’ and ‘JingZao 39’, segregating for stone and stoneless fruit.

QLTI-06. NEXT GENERATION SEQUENCING SIGNIFICANTLY IMPROVES THE DIAGNOSTIC ACCURACY IN GLIOBLASTOMAS

Abstract The 2021 WHO Classification of CNS Tumours recognizes the presence of TERT promoter (pTERT) mutations, combined +7/-10 chromosome copy-number alterations, or EGFR amplification as a defining molecular feature(s) of glioblastoma, independent of histology. We evaluated these biomarkers in a cohort of Indian patients using a single-run next-generation sequencing (NGS) platform at a relatively low depth of 100. Seventy-four adult diffuse gliomas were selected from the Exsegen Genomics Research Pvt Ltd Biobank established during the trial CTRI/2021/09/036861 after reviewing their clinical and pathological data. Paired surgical tissue and blood samples underwent whole-exome sequencing on an Illumina NovaSeq-6000 (Illumina, San Diego, USA). Single-nucleotide variations (SNVs) were processed using the DRAGEN pipeline to generate Mutation Annotation Format (MAF) files. Copy number variations (CNVs) analysis was performed using GATK and GISTIC2.0. Thirty-nine glioma samples with IDH1R132H mutations were excluded. A total of 32/35 (91.4%) IDH-wildtype tumours met the 2021 WHO criteria for glioblastoma, IDH-wildtype and were included. Twenty-four glioblastomas also underwent whole-transcriptome analysis. Microvascular proliferation (n=24, 75%) and necrosis (n=30, 93.9%) were present in the majority. pTERT mutations were identified in 23 (71.9%) samples, with C228T mutations (n=15/23, 65.2%) being more common than C250T mutations (n=8/23, 34.8%). EGFR amplification, defined as ≥5 copies by NGS CNV, was seen in 15 (46.9%) samples, with a strong correlation between EGFR CNV and logEGFR expression (r=0.79; p=0.017). Whole-arm +7/-10 genotype was evident in 17 (53.1%) tumours, and another 5 (15.6%) showed partial copy-number alterations. Notably, two gliomas without necrosis and proliferation initially diagnosed as oligodendrogliomas due to misinterpretation of the IDH immunostain, were reclassified as glioblastoma, IDH-wildtype. The data was validated using Sanger Sequencing and targeted panel sequencing to a depth of 500. Even low depth NGS sequencing, that is cost effective and time efficient offers significant improvements in diagnostic molecular neuropathology.

The discrepancy of somatic BRCA1/2 pathogenic variants from two different platforms in epithelial ovarian, fallopian tube, and peritoneal cancer

The somatic BRCA1 or BRCA2 Pathogenic Variant (PV)/Likely PV (LPV) from Next Generation Sequencing (NGS) is the most important biomarker for PARP inhibitor use and maintenance-targeted therapies. A discrepancy in the detection rates of BRCA1 and BRCA2 PV/LPV was identified among the NGS platforms. The objective of this study was to compare the somatic BRCA results from two distinct platforms using the same cohort and to identify the causes of these differences. Patients with epithelial ovarian cancer who concurrently underwent tumor NGS using two different platforms between January 2022 and June 2023 were included in this study. The two platforms used were in-house tumor NGS (Illumina NextSeq 550Dx, SureSelectXT library kit, and datasets from 1000 Genomes, ESP6500, ExAC, and ClinVar) and GreenPlan homologous recombination deficiency (HRD) test (Illumina NextSeq 550Dx, customized Twist Bioscience library kit, and datasets from COSMIC and OncoKB). The results of somatic mutations in BRCA1 and BRCA2 were compared between the two platforms. Of the 118 patients, 11.9% (n = 14) exhibited a discordant interpretation of BRCA1 or BRCA2 between the two platforms. Eleven patients (9.3%) exhibited negative results in the in-house platform but positive results (eight seven as PV of BRCA1, one as PV of BRCA2, one as LPV of BRCA1, and two as LPV of BRCA2) in the GreenPlan HRD test, while three patients (2.6%) had positive BRCA pathogenic variants (two as PV of BRCA1 [c.3340G > T, c.5152 + 3 A > C], one as LPV of BRCA2 [c.8174G > T], and one as LPV of BRCA1 [c.5017_5019delCAC]) in the in-house platform but a negative result in the GreenPlan HRD test. The discordance rate of somatic BRCA1 or BRCA2 mutations from different platforms was approximately 12%. In the case of the strong implication of BRCA PV/LPV with a negative result with one genetic test, different platforms could be considered in limited cases. Careful interpretation and further studies for the cross-validation of gene analysis platforms are needed.

Methodological approaches in 16S sequencing of female reproductive tract in fertility patients: a review.

The female genital tract microbiome has become a particular area of interest in improving assisted reproductive technology (ART) outcomes with the emergence of next-generation sequencing (NGS) technology. However, NGS assessment of microbiomes currently lacks uniformity and poses significant challenges for accurate and precise bacterial population representation. As multiple NGS platforms and assays have been developed in recent years for microbiome investigation-including the advent of long-read sequencing technologies-this work aimed to identify current trends and practices undertaken in female genital tract microbiome investigations. Areas like sample collection and transport, DNA extraction, 16S amplification vs. metagenomics, NGS library preparation, and bioinformatic analysis demonstrated a detrimental lack of uniformity. The lack of uniformity present is a significant limitation characterised by gap discrepancies in generation and interpretation of results. Minimal consistency was observed in primer design, DNA extraction techniques, sample transport, and bioinformatic analyses. With third-generation sequencing technology highlighted as a promising tool in microbiota-based research via full-length 16S rRNA sequencing, there is a desperate need for future studies to investigate and optimise methodological approaches of the genital tract microbiome to ensure better uniformity of methods and results interpretation to improve clinical impact.

Microbes and pathogens associated with shrimps - implications and review of possible control strategies.

Shrimp aquaculture has been growing rapidly over the last three decades. However, high-density aquaculture together with environmental degradation has led to increased incidence of shrimp infections. Thus, devising and implementing effective strategies to predict, diagnose and control the spread of infections of shrimps are crucial, also to ensure biosecurity and sustainability of the food industry. With the recent advancements in biotechnology, more attention has been given to develop novel promising therapeutic tools with potential to prevent disease occurrence and better manage shrimp health. Furthermore, owing to the advent of the next-generation sequencing (NGS) platforms, it has become possible to analyze the genetic basis of susceptibility or resistance of different stocks of shrimps to infections and how sustainable aquaculture could be made free of shrimp diseases.

Разработка кастомных баркодов для секвенирования на платформе MGI

The MGI (MGI Tech Co. Ltd., China) next-generation sequencing platform, including the DNBSEQ-G50, -G400, and -T7 sequencers, is being actively adopted in research. Despite its widespread adoption, challenges persist in the form of limitations associated with the manufacturer's provided barcode set for library preparation. These limitations include constraints on the number of samples that can be concurrently sequenced, compatibility issues with barcodes from diverse or incomplete sets, and restrictions on the sample ratio. Purpose: to develop a universal method that allows sequencing of up to 252 samples simultaneously on a single sequencer lane, while eliminating barcode-related limitations. We proposed a “quad method” that provides 4 or 4n+2 equilibration of barcodes. This paper also delves into its comprehensive analysis, verification procedures, seamless integration into the sequencing process and validation of the method on the DNBSEQ G-400 platform. The quad method showed efficiency and reliability, allowing sequencing of up to 252 samples simultaneously without compromising data quality. The proposed method optimizes library preparation and improves the flexibility of sequencing on the MGI platform.

Clinical and Genomic Characterization of ERBB2-Altered Gallbladder Cancer: Exploring Differences Between an American and a Chilean Cohort.

Gallbladder cancer (GBC) is a biliary tract malignancy characterized by its high lethality. Although the incidence of GBC is low in most countries, specific areas such as Chile display a high incidence. Our collaborative study sought to compare clinical and molecular features of GBC cohorts from Chile and the United States with a focus on ERBB2 alterations. Patients were accrued at Memorial Sloan Kettering Cancer Center (MSK) or the Pontificia Universidad Católica de Chile (PUC). Clinical information was retrieved from medical records. Genomic analysis was performed by the next-generation sequencing platform MSK-Integrated Mutation Profiling of Actionable Cancer Targets. A total of 260 patients with GBC were included, 237 from MSK and 23 from PUC. There were no significant differences in the clinical characteristics between the patients identified at MSK and at PUC except in terms of lithiasis prevalence which was significantly higher in the PUC cohort (85% v 44%; P = .0003). The prevalence of ERBB2 alterations was comparable between the two cohorts (15% v 9%; P = .42). Overall, ERBB2 alterations were present in 14% of patients (8% with ERBB2 amplification, 4% ERBB2 mutation, 1.5% concurrent amplification and mutation, and 0.4% ERBB2 fusion). Notably, patients with GBC that harbored ERBB2 alterations had better overall survival (OS) versus their ERBB2-wild type counterparts (22.3 months v 11.8 months; P = .024). The prevalence of lithiasis seems to be higher in Chilean versus US patients with GBC. A similar prevalence of ERBB2 alterations of overall 14% and better OS suggests that a proportion of them could benefit from human epidermal growth factor receptor type 2-targeted therapies. The smaller cohort of Chile, where the disease prevalence is higher, is a reminder and invitation for the need of more robust next-generation sequencing analyses globally.

Next Generation Sequencing and Genetic Analyses Reveal Factors Driving Evolution of Sweetpotato Viruses in Uganda.

Sweetpotato (Ipomoea batatas L.) is an essential food crop globally, especially for farmers facing resource limitations. Like other crops, sweetpotato cultivation faces significant production challenges due to viral infections. This study aimed to identify and characterize viruses affecting sweetpotato crops in Uganda, mostly those associated with sweetpotato virus disease (SPVD). Infected leaf samples were collected from farmers' fields in multiple districts spanning three regions in Uganda. MiSeq, a next-generation sequencing platform, was used to generate reads from the viral nucleic acid. The results revealed nine viruses infecting sweetpotato crops in Uganda, with most plants infected by multiple viral species. Sweet potato pakakuy and sweet potato symptomless virus_1 are reported in Uganda for the first time. Phylogenetic analyses demonstrated that some viruses have evolved to form new phylogroups, likely due to high mutations and recombination, particularly in the coat protein, P1 protein, cylindrical inclusion, and helper component proteinase regions of the potyvirus. The sweet potato virus C carried more codons under positive diversifying selection than the closely related sweet potato feathery mottle virus, particularly in the P1 gene. This study provides valuable insights into the viral species infecting sweetpotato crops, infection severity, and the evolution of sweet potato viruses in Uganda.

Enhanced heterologous gene expression in Trichoderma reesei by promoting multicopy integration

Trichoderma reesei displays a high capability to produce extracellular proteins and therefore is used as a platform for the expression of heterologous genes. In a previous study, an expression cassette with the constitutive tef1 promoter and the cbh1 terminator compatible with flow cytometry analysis was developed. Independent transformants obtained by a random integration into the genome of a circular plasmid containing the expression cassette showed a wide range of fluorescence levels. Whole genome sequencing was conducted on eight of the transformed strains using two next-generation sequencing (NGS) platforms: Illumina paired-end sequencing and Oxford Nanopore. In all strains, the expression plasmid was inserted at the same position in the genome, i.e., upstream of the tef1 gene, indicating an integration by homologous recombination. The different levels of fluorescence observed correspond to different copy numbers of the plasmid. Overall, the integration of a circular plasmid with the green fluorescence protein (egfp) transgene under the control of tef1 promoter favors multicopy integration and allows over-production of this heterologous protein on glucose. In conclusion, an expression system based on using the tef1 promotor could be one of the building blocks for improving high-value heterologous protein production by increasing the copy number of the encoding genes into the genome of the platform strain.Key points• Varied eGFP levels from tef1 promoter and cbh1 terminator expression.• Whole genome sequencing on short and long reads platforms reveals various plasmid copy numbers in strains.• Plasmids integrate at the same genomic site by homologous recombination in all strains.

Transforming Clinical Research: The Power of High-Throughput Omics Integration.

High-throughput omics technologies have dramatically changed biological research, providing unprecedented insights into the complexity of living systems. This review presents a comprehensive examination of the current landscape of high-throughput omics pipelines, covering key technologies, data integration techniques and their diverse applications. It looks at advances in next-generation sequencing, mass spectrometry and microarray platforms and highlights their contribution to data volume and precision. In addition, this review looks at the critical role of bioinformatics tools and statistical methods in managing the large datasets generated by these technologies. By integrating multi-omics data, researchers can gain a holistic understanding of biological systems, leading to the identification of new biomarkers and therapeutic targets, particularly in complex diseases such as cancer. The review also looks at the integration of omics data into electronic health records (EHRs) and the potential for cloud computing and big data analytics to improve data storage, analysis and sharing. Despite significant advances, there are still challenges such as data complexity, technical limitations and ethical issues. Future directions include the development of more sophisticated computational tools and the application of advanced machine learning techniques, which are critical for addressing the complexity and heterogeneity of omics datasets. This review aims to serve as a valuable resource for researchers and practitioners, highlighting the transformative potential of high-throughput omics technologies in advancing personalized medicine and improving clinical outcomes.

Total gonadotropin dose did not affect euploid blastocyst rates: an analysis of more than 19,000 oocytes.

To evaluate whether increasing total gonadotropin (Gn) dose is associated with changes in euploid blastocyst rate in preimplantation genetic testing (PGT) oocytes. This retrospective cohort study was conducted between 2017 and 2022, and 19,246 oocytes were grouped and analyzed based on tri-sectional quantiles of total Gn doses. Single reproductive medical center. All the patients who underwent PGT cycles, including PGT for aneuploidy, monogenic disorders, and structural rearrangements, were included. Next-generation sequencing platforms for chromosomal analysis. Blastocyst formation and euploid blastocyst rates. In total, 19,246 oocytes and 5375 PGT blastocysts were analyzed. There were significant differences in blastocyst formation and euploid blastocyst rates among the groups classified according to tri-sectional quantiles of total Gn doses. Significant differences in age, body mass index (BMI), proportion of primary infertility, anti-Müllerian hormone (AMH) levels, number of oocytes retrieved, controlled ovarian stimulation (COS) regimen, type of Gn, and PGT category were observed among the three groups. After stratifying the analysis by age, BMI, infertility diagnosis, AMH levels, number of oocytes retrieved, PGT category, type of Gn, and COS regimen, significant differences were only seen in a small number of specific subgroups. Furthermore, the results of the multiple logistic regression analysis showed that the blastocyst formation and euploid blastocyst rates did not significantly increase or decrease with the total Gn dose, whether treated as a continuous variable or divided into three Gn groups as categorical variables. Notably, advancing age was a risk factor for blastocyst formation and euploid blastocyst rates. PGT for structural rearrangements was a risk factor for blastocyst formation and euploid blastocyst rates as compared with PGT for aneuploidy. In the total PGT cycles, advancing age, and preimplantation genetic testing for structural rearrangements negatively affected blastocyst formation and euploid blastocyst rates; however, the total Gn dose did not affect blastocyst formation and euploid blastocyst rates.

Whole genome sequencing analysis of Mycobacterium tuberculosis reveals circulating strain types and drug-resistance mutations in the Philippines

The Philippines is a high-incidence country for tuberculosis, with the increasing prevalence of multi- (MDR-TB) and extensively-drug (XDR-TB) resistant Mycobacterium tuberculosis strains posing difficulties to disease control. Understanding the genetic diversity of circulating strains can provide insights into underlying drug resistance mutations and transmission dynamics, thereby assisting the design of diagnostic tools, including those using next generation sequencing (NGS) platforms. By analysing genome sequencing data of 732 isolates from Philippines drug-resistance survey collections spanning from 2011 to 2019, we found that the majority belonged to lineages L1 (531/732; 72.5%) and L4 (European-American; n = 174; 23.8%), with the Manila strain (L1.2.1.2.1) being the most prominent (475/531). Approximately two-thirds of isolates were found to be at least MDR-TB (483/732; 66.0%), and potential XDR-TB genotypic resistance was observed (3/732; 0.4%), highlighting an emerging problem in the country. Genotypic resistance was highly concordant with laboratory drug susceptibility testing. By finding isolates with (near-)identical genomic variation, five major clusters containing a total of 114 isolates were identified: all containing either L1 or L4 isolates with at least MDR-TB resistance and spanning multiple years of collection. Closer inspection of clusters revealed transmission in prisons, some involving isolates with XDR-TB, and mutations linked to third-line drug bedaquiline. We have also identified previously unreported mutations linked to resistance for isoniazid, rifampicin, ethambutol, and fluoroquinolones. Overall, this study provides important insights into the genetic diversity, transmission and circulating drug resistance mutations of M. tuberculosis in the Philippines, thereby informing clinical and surveillance decision-making, which is increasingly using NGS platforms.

Enhancing Precision in HIV Treatment: Validation of a Robust Next-Generation Sequencing System for Drug Resistance Mutation Analysis.

Multidrug-resistant HIV strains challenge treatment efficacy and increase mortality rates. Next-generation sequencing (NGS) technology swiftly detects variants, facilitating personalized antiretroviral therapy. This study aimed to validate the Vela Diagnostics NGS platform for HIV drug resistance mutation analysis, rigorously assessed with clinical samples and CAP proficiency testing controls previously analyzed by Sanger sequencing. The experimental approach involved the following: RNA extraction from clinical specimens, reverse transcription polymerase chain reaction (RT-PCR) utilizing the Sentosa SX 101 platform, library preparation with the Sentosa SQ HIV Genotyping Assay, template preparation, sequencing using the Sentosa SQ301 instrument, and subsequent data analysis employing the Sentosa SQ Suite and SQ Reporter software. Drug resistance profiles were interpreted using the Stanford HIV Drug Resistance Database (HIVdb) with the HXB2 reference sequence. The Vela NGS system successfully identified a comprehensive array of drug resistance mutations across the tested samples: 28 nucleoside reverse transcriptase inhibitors (NRTI), 25 non-nucleoside reverse transcriptase inhibitors (NNRTI), 25 protease inhibitors (PI), and 10 integrase gene-specific variants. Dilution experiments further validated the system's sensitivity, detecting drug resistance mutations even at viral loads lower than the recommended threshold (1000 copies/mL) set by Vela Diagnostics. This study underscores the validation and clinical applicability of the Vela NGS system, and its implementation may offer clinicians enhanced precision in therapeutic decision-making for individuals living with HIV.

Microbial Forensics: Comparison of MLVA Results According to NGS Methods, and Forensic DNA Analysis Using MLVA

Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying the origin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiological analysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, and advances in next generation sequencing(NGS) technology have enabled <i>in silico</i> anlysis of full-length whole genome sequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The United Nations Secretary-General’s Mechanism(UNSGM)’s external quality assessment exercise(EQAE) project, which we officially participated in 2023. We confirmed that the 3 unknown samples were <i>B. anthracis</i> through nucleic acid isolation and genetic sequence analysis studies. MLVA results on 32 loci of <i>B. anthracis</i> were analysed by using genome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing using short-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of the tandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy than NextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel error caused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 published MLVA databases of <i>B. anthracis</i>, and MLVA results of 10 strains were identical with 3 unkonwn samples. As a result of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as <i>B. anthracis</i> strain A4564 which is associated with injectional anthrax isolates in heroin users.

Investigation of Targeted Genes and Identification of Novel Variants with Next Generation Sequencing Method in Hearing Loss.

Hearing loss is a widespread condition throughout the world. It may affect patients from newborns to the elderly. There are too many reasons for hearing loss, including congenital hearing loss, virus infections, age-related situations, and traumatic situations, which may be related to the immune-mediated system. Fifty percent of hearing loss is related to genetic mutations and defects; genetic causes are highly heterogeneous, so the analysis of new variants are important for diagnosis. We aimed to describe the importance of detected gene variations by using targeted gene panels in the Next-Generation-Sequencing (NGS) platform. Eighty-one hearing loss targeted genes were investigated using Illumina NextSeq550 technology in 100 participants with hearing loss between 2017 and 2022 in our Genetic Diseases Evaluation Center. Targeted genes were performed on 100 patients with hearing loss diagnosis. The total number of detected variants was 77. Forty-seven cases have likely pathogenic/pathogenic variants. Thirty of them have uncertain clinical significance variants, and from the detected variants, 8 are novel. In this research, we highlighted that earlier detection of hearing loss using molecular genetic methods may help us understand the etiology and orient for a better prognosis. Results detected by using the NGS platform can assist and improve the diagnosis. In this study, the diagnostic rate with targeted genes was detected as 35.29%. It has an important role in clinical practice as the recommendation of cochlear implants. Clarifying the genotype and phenotype correlation helps us figure out the etiology of hearing loss and also the worth of genetic counseling in hereditary hearing loss.

Oxford nanopore sequencing-based assay for BTD gene screening: Design, clinical validation, and variant frequency assessment in the Turkish population

Biotinidase deficiency (BTD) is an autosomal recessive disorder characterized by impaired recycling of the water-soluble vitamin biotin which leads to a spectrum of clinical manifestations ranging from mild to severe, including mainly neurological and cutaneous symptoms. Biotin supplementation is a cornerstone of treatment, but diagnosis often relies on measuring serum enzyme activity, which needs to be confirmed by genetic analysis. Thus, molecular methods become necessary in the differential diagnosis of BTD. Accordingly, countries with a high-incidence have implemented next-generation sequencing (NGS) techniques to newborn screening programs for BT. Nevertheless, NGS platforms, while well-established, present challenges in cost, labor, accessibility, and duration for newborn screening programs targeting BTD, therefore these limitations necessitate the exploration of alternative systems to ensure efficient and widespread screening. Here, third-generation sequencing platforms, notably Oxford Nanopore Technology (ONT), present promising solutions to the associated challenges. Hence, in the present study, we aimed to develop an ONT-based assay for the screening of BTD gene. After designing and optimizing primers for long-PCR using reference DNA, we assessed the performance of the ONT assay in BTD patients previously diagnosed by enzyme assay and confirmed using Illumina-based sequencing. The results demonstrate a strong correlation between the two methods, indicating the reliability of the ONT-based assay. Moreover, this first in-house single gene testing specifically tailored for BTD successfully detected previously known genetic variants with high sequencing depths, affirming the effectiveness of ONT-based sequencing in human genetics.

Comparing Gold-Standard Sanger Sequencing with Two Next-Generation Sequencing Platforms of HIV-1 gp160 Single Genome Amplicons.

Our goal was to assess the accuracy of next generation sequencing (NGS) compared with Sanger. We performed single genome amplification (SGA) of HIV-1 gp160 on extracted tissue DNA from two HIV+ individuals. Amplicons (n = 30) were sequenced with Sanger or reamplified with barcoded primers and pooled before sequencing using Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PB). For each amplicon, a consensus sequence for NGS reads was obtained by (1) mapping reads to the Sanger sequence when available ("reference-based") or (2) mapping reads to a "pseudo-reference" sequence, i.e., a consensus sequence of a subset of NGS reads ("reference-free"). PB reads were clustered based on genetic similarity. A Sanger consensus sequence was obtained for 23/30 amplicons, for which all NGS consensus sequences were identical (n = 9) or nearly identical (n = 14) compared with Sanger. For the nine mismatches between Sanger/NGS, the nucleotide in the NGS sequence matched all other sequences from that patient. Of the 7/30 amplicons without a Sanger sequence, NGS sequences had ≥35 ambiguous calls in five amplicons and 0 ambiguitiesin two amplicons. Analysis of the electropherograms showed failure of a single sequencing primer for the latter two amplicons (consistent with a single template) and overlapping peaks for the other five (consistent with multiple templates). Clustering results closely followed the Sanger/NGS consensus results, where amplicons derived from a single template also had a single cluster and vice versa (with one exception, which could be the result of barcode misidentification). Representative sequences from the clusters contained 2-13 differences compared with Sanger/NGS. In summary, we show that both ONT and PB can produce amplicon consensus sequences with similar or higher accuracy compared with Sanger and, importantly, without the need for a known reference sequence. Clustering could be useful in some circumstances to predict or confirm the presence of multiple starting templates.

Non-Coding RNAs of Mitochondrial Origin: Roles in Cell Division and Implications in Cancer.

Non-coding RNAs (ncRNAs) are a heterogeneous group, in terms of structure and sequence length, consisting of RNA molecules that do not code for proteins. These ncRNAs have a central role in the regulation of gene expression and are virtually involved in every process analyzed, ensuring cellular homeostasis. Although, over the years, much research has focused on the characterization of non-coding transcripts of nuclear origin, improved bioinformatic tools and next-generation sequencing (NGS) platforms have allowed the identification of hundreds of ncRNAs transcribed from the mitochondrial genome (mt-ncRNA), including long non-coding RNA (lncRNA), circular RNA (circRNA), and microRNA (miR). Mt-ncRNAs have been described in diverse cellular processes such as mitochondrial proteome homeostasis and retrograde signaling; however, the function of the majority of mt-ncRNAs remains unknown. This review focuses on a subgroup of human mt-ncRNAs whose dysfunction is associated with both failures in cell cycle regulation, leading to defects in cell growth, cell proliferation, and apoptosis, and the development of tumor hallmarks, such as cell migration and metastasis formation, thus contributing to carcinogenesis and tumor development. Here we provide an overview of the mt-ncRNAs/cancer relationship that could help the future development of new biomedical applications in the field of oncology.

Molecular Assessment of Genetic Diversity and Bottleneck in Hair Goat Reared in Türkiye

Objective: Being the most preferred and geographically distributed in Türkiye, the Hair goat was screened at a molecular level to evaluate genetic diversity and population structure via microsatellite DNA markers. This paper also aimed to investigate the effects of genetic bottleneck to evaluate whether the Hair goat has maintained its effective population size in recent past. Material and Methods: A total of 411 Hair goats were sampled from farms participating in the "Hair Goat Breeding" project, initiated by the General Directorate of Agricultural Research and Policies in Aydın and Denizli provinces. Sampled animals were genotyped with 18 microsatellite loci to assess genetic diversity, population structure, and genetic bottleneck. Results: A total of 341 different alleles were observed across 18 microsatellite loci in which the highest number of alleles (26) and effective alleles (10.18) were detected in INRA005 and HSC loci, respectively. The average observed heterozygosity (0.73) was lower than the expected value (0.83), whereas all loci turned out to be highly informative (PIC>0.50). Factorial correspondence analysis separated animals into two groups, while a genetic admixture was detected between these groups. STRUCTURE analysis, on the other hand, confirmed that 411 animals were derived from three ancestral populations in which the third group is drawn due to admixed individuals. The Wilcoxon test and mode-shift indicator detected a lack of genetic bottleneck indicating that Hair goats reared in Türkiye have maintained their effective population size in recent past. Conclusion: This study validates that used microsatellite markers are highly polymorphic and could be utilized for revealing genetic diversity in different local goat breeds. The findings recovered in this study could be integrated into breeding and conservation programs, while further studies should adopt SNP array technologies and next-generation sequencing platforms to reveal deeper knowledge about the genetic diversity and population structure of Anatolian goat breeds.