Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome.
Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome.
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DOI：
10.1038/s41598-018-32615-8
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发表时间：
2018-09-26
影响因子：
4.6
通讯作者：
Barnes JR
中科院分区：
文献类型：
--
作者：
Keller MW;Rambo-Martin BL;Wilson MM;Ridenour CA;Shepard SS;Stark TJ;Neuhaus EB;Dugan VG;Wentworth DE;Barnes JR
RNA病毒的编码完整基因组首次以其原始形式被测序。此前，RNA 测序是通过放射性标记 RNA 的化学降解来进行的，这是一种只能产生短序列的困难方法。相反，RNA 通常通过将其复制到 cDNA 中来间接测序，而 cDNA 通常通过 PCR 扩增为 dsDNA，随后使用各种 DNA 测序方法进行分析。我们设计了一个接头来缩短甲型流感病毒基因组的高度保守末端，以将 (-) 正义 RNA 靶向至 Oxford Nanopore MinION 测序平台上的蛋白质纳米孔中。利用这种方法，从感染 rA/Puerto Rico/8/1934 (H1N1) 流感病毒的鸡蛋 (EID50 6.8 × 109) 的尿囊液中提取总 RNA，我们成功对编码完整甲型流感病毒基因组进行了测序，具有 100% 的核苷酸覆盖率、99% 的一致性以及 99% 的读取映射到甲型流感病毒。通过利用相同的方法，人们可以重新设计接头，以将靶标扩展到包括病毒 mRNA 和 (+) 有义 cRNA，它们对于病毒生命周期或其他病原体至关重要。这种方法还具有识别和量化剪接变体和碱基修饰的潜力，而目前的方法实际上无法测量这些剪接变体和碱基修饰。
For the first time, a coding complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabeled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza A virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method with total RNA extracted from the allantoic fluid of influenza rA/Puerto Rico/8/1934 (H1N1) virus infected chicken eggs (EID50 6.8 × 109), we demonstrate successful sequencing of the coding complete influenza A virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza A virus. By utilizing the same methodology one can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle, or other pathogens. This approach also has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.
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影响因子：
14.9
作者：
Ebhardt HA;Tsang HH;Dai DC;Liu Y;Bostan B;Fahlman RP
通讯作者：
Fahlman RP
影响因子：
3.7
作者：
Johnson A;Chen LM;Winne E;Santana W;Metcalfe MG;Mateu-Petit G;Ridenour C;Hossain MJ;Villanueva J;Zaki SR;Williams TL;Cox NJ;Barr JR;Donis RO
通讯作者：
Donis RO
影响因子：
5.2
作者：
Wang J;Mignon A;Snoeck D;Wiktor V;Van Vliergerghe S;Boon N;De Belie N
通讯作者：
De Belie N
影响因子：
64.8
作者：
LECLERC, J
通讯作者：
LECLERC, J
影响因子：
1.6
作者：
Ma, Jun;Liu, Kang;Cao, Yongchang
通讯作者：
Cao, Yongchang