3/25/2023 0 Comments Clc genomics workbench manualAlthough a variety of approaches and library preparation methods have been described for the sequencing of avian influenza viruses on the MinION platform (e.g., native barcoding kit (SQK-LSK109, PCR barcoding kit (SQK-PBK004), and rapid barcoding kit (SQK-RBK004) ), the performance differences between these methods have not yet been evaluated. Moreover, strategies to achieve >99% accuracy (often by increasing the depth of coverage, i.e., increasing the number of reads over each sequence location) in both SARS-CoV-2 and avian influenza virus sequencing have been published. Several studies have described the whole-genome amplification and nanopore sequencing of avian influenza viruses from both wild and domestic bird samples. Additionally, the smaller capacity of the MinION may be well-suited to the number of avian influenza surveillance samples that need to be sequenced at any one time by an individual diagnostic laboratory. For example, since 2021, in spite of nearly 2400 HPAI H5Nx outbreaks in poultry and over 2700 similar events in wild birds in Europe, plus another 673 detections in poultry and 4362 detections in wild birds in the United States, as of 8 December 2022, only 3116 HPAI H5Nx genomes were deposited in the GenBank and Global Initiative on Sharing All Influenza Data (GISAID) databases.īy producing readable sequencing data within 30 min, the MinION platform offers the ability to decrease the time for pathogen identification when speed is of the essence. To date, however, there are no global programs for the large-scale sequencing and analysis of HPAI viral sequences analogous to that for SARS-CoV-2. Whole-genome sequencing, in conjunction with epidemiological information, has often been used to elucidate routes of introduction and patterns of spread in HPAI outbreaks. In recent years, highly pathogenic avian influenza (HPAI) viruses have been introduced and have circulated in wild birds and poultry in Asia, Europe, and North America. With over four million genomes sequenced, patterns of introduction, dissemination, and evolution of variants have been well-described, but one area where the state of the art might improve is the ability to sequence in real time or near real time. The emergence and global spread of SARS-CoV-2 has led to widespread use of whole-genome sequencing to guide therapeutic countermeasures for individuals, as well as for population-scale epidemiological monitoring. ![]() ![]() A limited amplification procedure and a rapid procedure are found to be best among the approaches taken. ![]() In this study, we evaluate different primer sets for the whole genome amplification of influenza A virus and evaluate five different library preparation approaches for sequencing on the nanopore platform using the MinION flow cell. We evaluate this platform for routine use in a diagnostic laboratory. This platform offers the potential to extend in-house sequencing capacities to laboratories that may otherwise lack resources to adopt sequencing technologies requiring large benchtop instruments. To facilitate this objective, we investigate a next-generation sequencing platform that uses a portable nanopore sequencing device to generate and present data in real time. The recent rapid and extensive introduction and spread of highly pathogenic avian influenza virus in Europe, North America, and elsewhere raises the need for similarly rapid sequencing to aid in appropriate response and mitigation activities. As exemplified by the global response to the SARS-CoV-2 pandemic, whole-genome sequencing played an important role in monitoring the evolution of novel viral variants and provided guidance on potential antiviral treatments.
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