A nanopore sequencing scheme for MiniHap markers and its application in kinship analysis.

Evaluation of microhaplotype panels for complex kinship analysis using massively parallel sequencing

Nanopore sequencing of MiniHap biomarkers for forensic DNA mixture deconvolution: A proof-of-principle study.

BackgroundDongxiang group, as an important minority, resides in Gansu province which is located at the northwest China, forensic detection system with more loci needed to be studied to improve the application efficiency of forensic case investigation in this group.MethodsA 60-plex system including 57 autosomal deletion/insertion polymorphisms (A-DIPs), 2 Y chromosome DIPs (Y-DIPs) and the sex determination locus (Amelogenin) was explored to evaluate the forensic application efficiencies of individual discrimination, kinship analysis and biogeographic origin prediction in Gansu Dongxiang group based on the 60-plex genotype results of 233 unrelated Dongxiang individuals. The 60-plex genotype results of 4582 unrelated individuals from 33 reference populations in five different continents were also collected to analyze the genetic background of Dongxiang group and its genetic relationships with other continental populations.ResultsThe system showed high individual discrimination power, as the cumulative power of discrimination (CPD), cumulative power of exclusion (CPE) for trio and cumulative match probability (CMP) values were 0.99999999999999999999997297, 0.999980 and 2.7029E− 24, respectively. The system could distinguish 98.12%, 93.78%, 82.18%, 62.35% and 39.32% of full sibling pairs from unrelated individual pairs, when the likelihood ratio (LR) limits were set as 1, 10, 100, 1000 and 10,000 based on the simulated family samples, respectively. Additionally, Dongxiang group had the close genetic distances with populations in East Asia, especially showed the intimate genetic relationships with Chinese Han populations, which were concluded from the genetic affinities and genetic background analyses of Dongxiang group and 33 reference populations. In terms of the effectiveness of biogeographic origin inference, different artificial intelligent algorithms possessed different efficacies. Among them, the random forest (RF) and extreme gradient boosting (XGBoost) algorithm models could accurately predict the biogeographic origins of 99.7% and 90.59% of three and five continental individuals, respectively.ConclusionThis 60-plex system had good performance for individual discrimination, kinship analysis and biogeographic origin prediction in Dongxiang group, which could be used as a powerful tool for case investigation.

A proof-of-principle study: The potential application of MiniHap biomarkers in ancestry inference based on the QNome nanopore sequencing

Next-generation sequencing of the metagenome (mNGS) is gaining traction as a valuable tool for diagnosing infectious diseases. Compared to mNGS, pathogen detection based on Oxford Nanopore Technology further shortens the detection time. This study seeks to assess the efficacy of Nanopore sequencing in identifying pathogens associated with community-acquired pneumonia (CAP) among elderly individuals in China. From January 2023 to June 2023, elderly patients with CAP were prospectively recruited from Hangzhou First People's Hospital. A comprehensive set of clinical data was gathered, and bronchoalveolar lavage (BAL) fluid samples were collected. Concurrently, pathogen identification was performed using conventional microbiological diagnostic methods, Illumina sequencing, and Nanopore sequencing, and the diagnostic efficacy of pathogen detection was compared. The study included a total of 29 patients. The diagnostic positivity rates of traditional microbiological detection, Illumina sequencing, and Nanopore sequencing were 24.1%, 51.7%, and 48.3%, respectively. Their diagnostic specificities were 91.7%, 50%, and 75%, respectively. Compared to traditional microbiological detection, both Nanopore and Illumina sequencing showed significantly increased sensitivity. However, Nanopore sequencing exhibited relatively better consistency with the final clinical comprehensive diagnosis, with a Kappa value of 0.574. This outperformed traditional microbiological detection and Illumina sequencing, which had a Kappa value of 0.296 and 0.402, respectively. In addition, Nanopore sequencing required the shortest turnaround time. Nanopore sequencing technology demonstrates as a reliable and rapid method for detecting pathogens in elderly patients with CAP.

BackgroundMicrobial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis.MethodsUsing full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix™ SK-2S, Sugi® Eyespear, Cotton, Rayon, Dryswab™, Hydraflock®, Albumin-coated, Purflock®, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods.ResultsWe found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (p < 0.001), with the Sugi® Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results.ConclusionWe have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing.

It is challenging to distinguish embryos with a balanced translocation karyotype from a normal karyotype by existing conventional genetic testing methods. However, in germ-cell gamete generation, chromosome exchange and separation through cell meiosis form a different proportion of unbalanced gametes. Adverse birth events may occur, such as repeated miscarriages and fetal birth defects. In this study, the exact breakpoints of structural variation (SV) from two balanced translocation carrier families by using Nanopore long reads sequencing technology were obtained, and haplotype analysis and Sanger verified the accuracy of the detection results, confirming the application value of the Nanopore sequencing technology in the detection of balanced translocation before embryo implantation. Nanopore long-read sequencing was performed to find the precise breakpoint of chromosome-balanced translocation carriers. The breakpoints were subsequently verified by designing primers across the breakpoints and Sanger sequencing. Haplotype linkage analysis of SNPs which can be linked by a read block of families around the breakpoint regions was followed. After frozen (-thawed) embryo transfer (FET), prenatal cytogenetic analysis of amniotic fluid cells confirmed the predicted karyotypes from the transferred embryos. The presence of breakpoints was detected in three embryos of patient 1. No breakpoints were detected in either embryo of patient 2. One balanced translocated embryo from patient 1 and one normal euploid embryo from patient 2 were transplanted back into the patients, and amniotic fluid cells were analyzed for the karyotype of fetuses. The results were entirely consistent with the fetal karyotype. And through late follow-up, both patients successfully had a live birth fetus. The breakpoint location of the balanced chromosome translocation can be accurately found by Nanopore sequencing. The haplotype of carriers can be successfully constructed by Nanopore and sanger sequencing confirmed that the results were accurate. This is very advantageous for preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) detection in the families without proband.

Much tremendous break through have been obtained in recent years for nanopore sequencing to achieve the goal of $1000 genome. As a method of single molecule sequencing, nanopore sequencing can discriminate the individual molecules of the target DNA strand rapidly due to the current blockages by translocating the nucleotides through a nano-scale pore. Both the protein-pores and solid-state nanopore channels which called single nanopore sequencing have been studied widely for the application of nanopore sequencing technology. This review will give a detail representation to protein nanopore and solid-state nanopore sequencing. For protein nanopore sequencing technology, we will introduce different nanopore types, device assembly and some challenges still exist at present. We will focus on more research fields for solid-state nanopore sequencing in terms of materials, device assembly, fabricated methods, translocation process and some specific challenges. The review also covers some of the technical advances in the union nanopore sequencing, which include nanopore sequencing combine with exonuclease, hybridization, synthesis and design polymer.

Introduction: Integration of human papillomavirus (HPV) DNA into the human genome is considered as a key event in cervical carcinogenesis. Different methods have been employed in exploring HPV integration sites, such as APOT, DIPS-PCR, and NGS. However, these methods have their drawbacks. APOT heavily relies on the quality of the sample due to its dependence on RNA, while DIPS-PCR mostly identifies known sites and has limited capability in uncovering new integration sites. Although the detection sensitivity of virus infection status increased significantly through the Illumina sequencing platform, there were still disadvantages remain for further improvement, including the detection accuracy and the complex integrated genome structure identification, etc. Also, it requires large amounts of sequencing data, and thus is not applicable in clinical usage, which requires fast and accurate results. Therefore, the development of a new method for HPV integration detection with high accuracy and prompt reporting capacity is crucial for future clinical application. Methods: We established a novel library preparation strategy for the detection of HPV integration in the human genome using Oxford Nanopore sequencing. Using this novel technology, Nanopore libraries were prepared from HPV positive cell lines HeLa and SiHa followed by sequencing on the Oxford Nanopore. We also determined HPV integration sites in five patients with cervical cancer patients with HPV positive tumors using Ilumina WGS. HPV integrations and breakpoints in human genome from both Nanopore and Ilumina WGS were extracted using Minimap and ViFi tool respectively. All HPV integration sites from cell lines and patient samples were validated using Sanger sequencing. Results: The novel library preparation approach for detection of HPV integrations with Nanopore sequencing technology were sensitive and efficient in detection of commonly reported HPV breakpoints in HPV positive cell lines HeLa and SiHa. Additionally, we applied this method to clinical samples from five patients subjected to Whole Genome Sequencing, consistently yielding concordant results. Conclusion: Our Nanopore based assay for detecting HPV integrations without the need for WGS demonstrated high efficacy. These data highlight the sensitivity and efficiency of this methodology in detecting HPV integrations events, emphasizing its potential as a reliable diagnostic tool in understanding pathophysiology of HPV-related malignancies and its use as prognostic markers in clinical settings. Citation Format: Preetiparna Parida, Nivedita Mukherjee, Agastya Singh, Ashima Singh, Krishna Sharan, Mahadev Rao, Shirley Lewis, Sabarinathan Radhakrishnan, Rama Rao Damerla. Accurate detection of human papillomavirus breakpoints in cervical cancers using a novel library preparation strategy for nanopore sequencing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2937.

To review the principles and application of Nanopore Sequencing Technology (NPST) in oral cancer. Oral cancer is a disease caused by aberrations in the genes. Substantial research at the genomic level is still required for in-depth understanding of the molecular mechanism in oral cancer. The advent of the novel nanopore sequencing technique has the potential to detect the alterations at the genomic level. This review highlights nanopore sequencing, its advantages and disadvantages, and how research supports its application in the field of oral oncology. Web-based search via PubMed database, internet sources using keywords "nanopore sequencing, third-generation sequencing, next generation sequencing, cancer, oral squamous cell carcinoma, genetic, epigenetic, oncogenic viruses" was performed in this review. Original research, reviews, and short discussions published from 2008 to 2020 were included. The findings are discussed with emphasis on common gene mutations, epigenetic alterations, and oncogenic viruses in oral cancer. A brief mention regarding translational nanopore sequencing research in oral cancer and future perspectives is also discussed. The results obtained reveal that cost-effectiveness and rapid turnaround time make nanopore sequencing an enticing platform to resolve the ambiguity of genomes, epigenomes, and transcriptomes. The findings will encourage researchers to further adopt NPST in their studies and give an overview of the latest findings of oral squamous cell carcinoma (OSCC) management. To highlight the importance of NPST application in OSCC studies, this paper not only discusses the use of NPST in identifying the behavior of malignancy but also implies the need for further research using this technique. The review suggests that nanopore sequencing can be utilized for diagnosis and achieving personalized treatment in each oral cancer patient.

Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME (Oxford Nanopore Technologies—ONT, Oxford, UK) and an in-house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 (Oxford Nanopore Technologies—ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies—ONT, Oxford, UK) were compared. In conclusion, the current study shows that the nanopore sequencing platform is comparable with the Illumina platform in detection bacterial genera of the nasal microbiota, but the nanopore platform does have problems in detecting bacteria within the genus Corynebacterium. Although advances are being made, thorough validation of the nanopore platform is still recommendable.

Nanopore sequencing technology (NST), also known as single molecule real-time sequencing technology, allows deciphering single DNA and RNA molecules without polymerase chain reaction (PCR). Currently, NST has undergone rapid development in scientific research and clinical practice, with substantial improvements made in the read length and sequencing throughput. These breakthroughs have required extensive development of experiments and bioinformatics methods to take full advantage of nanopore long-read sequencing in areas such as genomics, transcriptomics, epigenomics and epitranscriptomics [1,2]. NST is currently being applied to genome assembly, full-length transcript sequencing and basemodification detection, and other specialized fields, such as rapid clinical diagnosis of pathogenic infections [3] and detection of pathogenic variants [4].However, due to the designed principle and data characteristics of NST [5], bioinformatics methods and tools developed based on NST have not been entirely satisfactory in terms of their adaptability, accuracy, and robustness. Analytical tools for next-generation sequencing (NGS) cannot be directly used to analyze NST data, so there is an urgent need for specially designed and efficient NST bioinformatics analysis methods and toolkits to provide solutions for scientific research and clinical practice. This Research Topic includes four papers that explain the application of NST and related bioinformatics analysis tools from different perspectives, such as the Android App (KARGAMobile) for real-time portable analysis of KARGAMobile uses a compressed ARG reference database and different internal data structures to save RAM usage, and has a user-friendly graphical interface that guides file browsing, loading, parameterization, and process execution. The output files are post-processed to create visual, printable, and shareable reports that help users interpret the results. Sun et al. used TCGA and GTEx data to identify KIF20A as a hub gene for lung adenocarcinoma with comprehensive bioinformatics analysis. They found a negative correlation between KIF20A expression and overall survival, progression-free survival, and disease-free survival. They also found that KIF20A knockdown inhibited cell proliferation, induced G2/M phase arrest, and promoted apoptosis. Moreover, they speculated that KIF20A may be used as a prognostic biomarker and therapeutic target for LUAD.Lang et al. developed NanoSTR, a method for target short tandem repeats detection based on NST.NanoSTR uses statistical methods such as "multi-sampling" and Length-Number-Rank (LNR) information with sequencing data to detect the target STR locus and genotyping. Compared with existing STR detection tools, NanoSTR showed higher accuracy, better performance, and certain robustness.NanoSTR not only avoided the problem of genotyping errors or inability caused by the NST data characteristics, but also eliminated the requirement of a genome background database construction or reference genome alignment, which could reduce the consumption of computing resources, and did not require secondary processing.Chen et al. developed several data augmentation strategies for NST to reduce the size requirements of the dataset while improving the robustness, accuracy, and other performance of the basecalling algorithm. These data augmentation strategies could improve the accuracy of basecalling by more than 1% without doubling the size of the training dataset. This work not only reduced the time and computational costs consumed during development but also provided new insights into the new basecalling algorithm.In conclusion, although there are not many papers in our research topic, we believe that these papers can provide help and reference to similar research fields, and we hope to promote the application of NST in more scientific research and practical scenarios.

Our research objective was to evaluate the genetic parameters in the populations of Akhal-Teke horses in 4 countries: Czech Republic, Russia, Estonia and Switzerland. The experiment involved a total of 325 Akhal-Teke horses; 121 horses came from the Czech Republic, 152 were from Russia, 28 were from Estonia and 24 horses came from Switzerland. For the divided database of micro satellites we evaluated the following parameters: effective number of alleles, frequency of alleles for the groups of horses; the observed heterozygosity (HO); the expected heterozygosity (HE); the inbreeding coefficient (Fis); and the genetic distance. The researched population is polymorphous. The population in the Czech Republic differs from the other three countries in the numbers of alleles per locus. The Czech population also includes Akhal-Teke horses which are not purebred Akhal-Teke horses. A confirmation of this fact is the effective number of alleles. The population in the Czech Republic exhibits the highest mean number of effective alleles. The Akhal-Teke population in Estonia exhibits the highest mean observed heterozygosity. By contrast, the population in the Czech Republic exhibits the lowest mean observed heterozygosity. In the Czech Republic the mean Fis value is a positive number indicating a reduced number of heterozygotes in the Czech Akhal-Teke population. The genetic distance is the highest between populations of horses bred in Russia and Estonia. The genetic distance is the lowest between populations of Akhal-Teke horses bred in Russia and in the Czech Republic.

Alternaria alternata is an economically important pathogen that can cause severe plant diseases in various crops, such as citrus, pear, strawberry, and tobacco. Here, we report on the genome sequence of A. alternata DZ causing tobacco brown spot disease in most tobacco-producing areas. A total of 11 contigs, including 10 essential chromosomes and 1 mitochondria genome, were assembled by combining Oxford Nanopore and Illumina sequencing technologies. All 10 assembled chromosomes enabled detection of at least one of the telomeric terminal repeat sequences CCCTAA/TTAGGG. The assembled chromosomes were a total of 34.11 Mb in length with a GC content of 50.95%, whereas the mitochondrial genome was 50,595 bp in length with a GC content of 29.16%. We were unable to identify any evidence of conditionally dispensable chromosomes in the genome of A. alternata DZ, nor were we able to locate any ACT gene orthologs. This genome resource will provide valuable insights for comparative genomics research of the Alternaria genus and serve as a useful resource in investigations of plant-pathogen molecular interactions. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

BackgroundThis study aimed to evaluate the diagnostic efficacy of nanopore sequencing for Mycobacterium tuberculosis (MTB) drug resistance in respiratory specimens from pulmonary tuberculosis (PTB) patients. It compared it to the Xpert MTB/RIF and fluorescent polymerase chain reaction (PCR) melting curve to explore the validity and feasibility of detecting MTB drug resistance in respiratory specimens.MethodsThis study retrospectively analyzed 52 respiratory specimens. The proportional method applied the phenotypic drug susceptibility test (pDST) to respiratory specimens. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), consistency statistic (kappa) with phenotypic drug susceptibility testing (pDST), and the area under the curve (AUC) from the receiver operating characteristic (ROC) curve were calculated for nanopore sequencing, Xpert MTB/RIF, and fluorescent PCR melting curve. These calculations used the pDST results as the reference standard.ResultsAmong the resistance mutation genes detected by nanopore sequencing, rpoB, and katG were the most frequent, followed by embB, rpsL, gyrA, inhA, ahpC, gyrB, gid, and rrs. In bronchoalveolar lavage fluid (BALF) specimens, nanopore sequencing showed high sensitivity (100.00%,90.32%,82.35%,82.35%,100.00%,76.92%), specificity (70.00%,81.82%,88.00%,96.00%93.75%,93.10%0.100.00%), and AUC values (0.85,0.86,0.85, 0.89,0.97,0.85) for rifampicin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (SM), levofloxacin (LFX), moxifloxacin (MFX). Nanopore sequencing exhibited good detection efficacy (kappa value ≥ 0.70) and perfect diagnostic resistance value (AUC value ≥ 0.85). For RIF, nanopore sequencing showed Kappa values of 0.01 and 0.38 and AUC values of 0.02 and 0.18 higher than the Xpert MTB/RIF and fluorescent PCR melting curve, respectively; for INH, nanopore sequencing had a higher Kappa value of 0.65 and a higher AUC value of 0.32 than the fluorescent PCR melting curve. Nanopore sequencing provided superior overall performance.ConclusionNanopore sequencing has significant technical advantages and clinical application potential in detecting MTB drug resistance. Its rapid and highly accurate detection capabilities support early diagnosis and personalized treatment of drug-resistant MTB. As the technology continues to mature and the cost is further reduced, it is expected that nanopore sequencing technology will play a more important role in MTB resistance detection.