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Nano2NGS-Muta: a framework for converting nanopore sequencing data to NGS-liked sequencing data for hotspot mutation detection.

ABSTRACT: Nanopore sequencing, also known as single-molecule real-time sequencing, is a third/fourth generation sequencing technology that enables deciphering single DNA/RNA molecules without the polymerase chain reaction. Although nanopore sequencing has made significant progress in scientific research and clinical practice, its application has been limited compared with next-generation sequencing (NGS) due to specific design principle and data characteristics, especially in hotspot mutation detection. Therefore, we developed Nano2NGS-Muta as a data analysis framework for hotspot mutation detection based on long reads from nanopore sequencing. Nano2NGS-Muta is characterized by applying nanopore sequencing data to NGS-liked data analysis pipelines. Long reads can be converted into short reads and then processed through existing NGS analysis pipelines in combination with statistical methods for hotspot mutation detection. Nano2NGS-Muta not only effectively avoids false positive/negative results caused by non-random errors and unexpected insertions-deletions (indels) of nanopore sequencing data, improves the detection accuracy of hotspot mutations compared to conventional nanopore sequencing data analysis algorithms but also breaks the barriers of data analysis methods between short-read sequencing and long-read sequencing. We hope Nano2NGS-Muta can serves as a reference method for nanopore sequencing data and promotes higher application scope of nanopore sequencing technology in scientific research and clinical practice.

SUBMITTER: Lang J

PROVIDER: S-EPMC9022462 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Radical new possibilities of improved treatment of cancer are on offer from an advanced medical technology already demonstrating its significance: next-generation sequencing (NGS). This refined testing provides unprecedentedly precise diagnoses and permits the use of focused and highly personalized treatments. However, across regions globally, many cancer patients will continue to be denied the benefits of NGS as long as some of the yawning gaps in its implementation remain unattended. The challenges at the regional and national levels are linked because putting the solutions into effect is highly dependent on cooperation between regional- and national-level cooperation, which could be hindered by shortfalls in interpretation or understanding. The aim of the paper was to define and explore the necessary conditions for NGS and make recommendations for effective implementation based on extensive exchanges with policy makers and stakeholders. As a result, the European Alliance for Personalised Medicine (EAPM) developed a maturity framework structured around demand-side and supply-side issues to enable interested stakeholders in different countries to self-evaluate according to a common matrix. A questionnaire was designed to identify the current status of NGS implementation, and it was submitted to different experts in different institutions globally. This revealed significant variability in the different aspects of NGS uptake. Within different regions globally, to ensure those conditions are right, this can be improved by linking efforts made at the national level, where patients have needs and where care is delivered, and at the global level, where major policy initiatives in the health field are underway or in preparation, many of which offer direct or indirect pathways for building those conditions. In addition, in a period when consensus is still incomplete and catching up is needed at a political level to ensure rational allocation of resources-even within individual countries-to enable the best ways to make the necessary provisions for NGS, a key recommendation is to examine where closer links between national and regional actions could complement, support, and mutually reinforce efforts to improve the situation for patients.

Project description:BACKGROUND:Next generation sequencing (NGS) is becoming widely used among diagnostics and research laboratories, and nowadays it is applied to a variety of disciplines, including veterinary virology. The NGS workflow comprises several steps, namely sample processing, library preparation, sequencing and primary/secondary/tertiary bioinformatics (BI) analyses. The latter is constituted by a complex process extremely difficult to standardize, due to the variety of tools and metrics available. Thus, it is of the utmost importance to assess the comparability of results obtained through different methods and in different laboratories. To achieve this goal, we have organized a proficiency test focused on the bioinformatics components for the generation of complete genome sequences of salmonid rhabdoviruses. METHODS:Three partners, that performed virus sequencing using different commercial library preparation kits and NGS platforms, gathered together and shared with each other 75 raw datasets which were analyzed separately by the participants to produce a consensus sequence according to their own bioinformatics pipeline. Results were then compared to highlight discrepancies, and a subset of inconsistencies were investigated more in detail. RESULTS:In total, we observed 526 discrepancies, of which 39.5% were located at genome termini, 14.1% at intergenic regions and 46.4% at coding regions. Among these, 10 SNPs and 99 indels caused changes in the protein products. Overall reproducibility was 99.94%. Based on the analysis of a subset of inconsistencies investigated more in-depth, manual curation appeared the most critical step affecting sequence comparability, suggesting that the harmonization of this phase is crucial to obtain comparable results. The analysis of a calibrator sample allowed assessing BI accuracy, being 99.983%. CONCLUSIONS:We demonstrated the applicability and the usefulness of BI proficiency testing to assure the quality of NGS data, and recommend a wider implementation of such exercises to guarantee sequence data uniformity among different virology laboratories.

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Nano2NGS-Muta: a framework for converting nanopore sequencing data to NGS-liked sequencing data for hotspot mutation detection.

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