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用于甲型流感纳米孔测序的定制条形码引物:性能增强且制备时间缩短。

Custom barcoded primers for influenza A nanopore sequencing: enhanced performance with reduced preparation time.

作者信息

Goraichuk Iryna V, Suarez David L

机构信息

Southeast Poultry Research Laboratory, U.S. National Poultry Research Center, Agriculture Research Service, U.S Department of Agriculture, Athens, GA, United States.

出版信息

Front Cell Infect Microbiol. 2025 Apr 15;15:1545032. doi: 10.3389/fcimb.2025.1545032. eCollection 2025.

Abstract

Highly pathogenic avian influenza is endemic and widespread in wild birds and is causing major outbreaks in poultry worldwide and in U.S. dairy cows, with several recent human cases, highlighting the need for reliable and rapid sequencing to track mutations that may facilitate viral replication in different hosts. SNP analysis is a useful molecular epidemiology tool to track outbreaks, but it requires accurate whole-genome sequencing (WGS) with sufficient read depth across all eight segments. In outbreak situations, where timely data is critical for controlling the spread of the virus, reducing sequencing preparation time while maintaining high-quality standards is particularly important. In this study, we optimized a custom barcoded primer strategy for influenza A whole-genome sequencing on the nanopore sequencing platform, combining the high performance of the Native Barcoding Kit with the prompt preparation time of the Rapid Barcoding Kit. Custom barcoded primers were designed to perform barcode attachment during RT-PCR amplification, eliminating the need for separate barcoding and clean-up steps, thus reducing library preparation time. We compared the performance of the custom barcoded primer method with the Native and Rapid barcoding kits in terms of read quality, read depth, and sequencing output. The results show that the custom barcoded primers provided performance comparable to the Native Barcoding Kit while reducing library preparation time by 2.3X compared to the Native kit and being only 15 minutes longer than the Rapid kit with better depth of sequencing. Additionally, the custom barcoded primer method was evaluated on a variety of clinical sample types. This approach offers a promising solution for influenza A sequencing, providing both high throughput and time efficiency, which significantly improves the time-to-result turnaround, making sequencing more accessible for real-time surveillance.

摘要

高致病性禽流感在野生鸟类中呈地方性流行且广泛传播,正在全球范围内的家禽以及美国奶牛中引发重大疫情,近期还出现了几例人类感染病例,这凸显了进行可靠且快速测序以追踪可能促进病毒在不同宿主中复制的突变的必要性。单核苷酸多态性(SNP)分析是追踪疫情的一种有用的分子流行病学工具,但它需要对所有八个基因片段进行具有足够读长深度的准确全基因组测序(WGS)。在疫情爆发的情况下,及时的数据对于控制病毒传播至关重要,在保持高质量标准的同时减少测序准备时间尤为重要。在本研究中,我们在纳米孔测序平台上优化了一种用于甲型流感全基因组测序的定制条形码引物策略,将原生条形码试剂盒的高性能与快速条形码试剂盒的快速准备时间相结合。定制条形码引物设计用于在逆转录聚合酶链反应(RT-PCR)扩增过程中进行条形码连接,无需单独的条形码标记和纯化步骤,从而减少文库制备时间。我们在读取质量、读取深度和测序产量方面比较了定制条形码引物方法与原生和快速条形码试剂盒的性能。结果表明,定制条形码引物的性能与原生条形码试剂盒相当,同时与原生试剂盒相比,文库制备时间减少了2.3倍,且比快速试剂盒仅长15分钟,但测序深度更好。此外,还对多种临床样本类型评估了定制条形码引物方法。这种方法为甲型流感测序提供了一种有前景的解决方案,兼具高通量和时间效率,显著缩短了获得结果的周转时间,使测序更便于用于实时监测。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/491b/12037546/a2e62bcd9c09/fcimb-15-1545032-g001.jpg

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