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首页> 外文期刊>Journal of Virological Methods >Comparison of nucleic acid extraction methods for next-generation sequencing of avian influenza A virus from ferret respiratory samples
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Comparison of nucleic acid extraction methods for next-generation sequencing of avian influenza A virus from ferret respiratory samples

机译：禽流感核酸提取方法对禽流验样品的下一代测序核酸提取方法

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摘要

Influenza A virus is a negative-sense RNA virus with a segmented genome consisting of eight RNA segments. Avian influenza A virus (AIV) primarily infects avian hosts and sporadically infects mammals, which can lead to adaptation to new species. Next-generation sequencing (NGS) of emerging AIV genomes extracted from respiratory samples collected on sequential days from animal models and clinical patients enables analysis of the emergence of evolutionary variants within the virus population over time. However, obtaining codon complete MV genome at a sufficient coverage depth for nucleotide variant calling remains a challenge, especially from post-inoculation respiratory samples collected at late time points that have low viral titers. In this study, nasal wash samples from ferrets inoculated with different subtypes of AIV were collected on various days post-inoculation. Each nasal wash sample was aliquoted and extracted using five commercially available nucleic acid extraction methods. Extracted influenza virus RNA was amplified and NGS conducted using Illumina Mi-Seq. For each nasal wash sample, completeness of AIV genome segments and coverage depth were compared among five extraction methods. Nucleic acids extracted by MagNA pure compact RNA isolation consistently yielded codon complete sequences for all eight genome segments at the required coverage depth at each time point sampled. The study revealed that DNase treatment was critical to the amplification of influenza genome segments and the downstream success of codon complete NGS from nasal wash samples. The findings from this study can be applied to improve NGS of influenza and other RNA viruses that infect the respiratory tract and are collected from respiratory samples.
机译：流感病毒是一种负面感测RNA病毒，其分段基因组由8个RNA段组成。禽流感的病毒（AIV）主要感染禽宿主并偶尔感染哺乳动物，这可能导致适应新物种。从动物模型和临床患者从动物模型和临床患者收集的呼吸样品中提取的新一代测序（NGS）从动物模型和临床患者中提取的呼吸样品中提取的呼吸样品中的呼吸样品中的序列能够随着时间的推移分析病毒群体内的进化变体的出现。然而，在足够的核苷酸变体调用的足够覆盖深度下获得密码子完成MV基因组仍然是一个挑战，尤其是在具有低病毒滴度的后期收集的接种后呼吸样本。在本研究中，在接种后的各个天中收集来自AIV不同AIV亚型不同亚型的雪貂的鼻腔样品。每个鼻腔洗涤样品等来使用五种市售核酸提取方法等分并提取。提取的流感病毒RNA被扩增，使用Illumina Mi-Seq进行的NGS。对于每个鼻腔洗涤样品，在五种提取方法中比较了AIV基因组区段的完整性和覆盖深度。通过MANGA纯致密的RNA分离提取的核酸始终如一地产生密码子完成所有八个基因组区段，在每个时间点采样时所需的覆盖深度。该研究表明，DNase治疗对流感基因组区段的扩增至关重要，以及密码子的下游成功从鼻腔洗涤样品完成NGS。本研究的发现可以应用于改善感染呼吸道的其他RNA病毒并从呼吸样品中收集的流感和其他RNA病毒。

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