RNA-seq accuracy and reproducibility for the mapping and quantification of influenza defective viral genomes.
RNA-seq accuracy and reproducibility for the mapping and quantification of influenza defective viral genomes.
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DOI：
10.1261/rna.077529.120
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发表时间：
2020-12
期刊：
影响因子：
--
通讯作者：
Naffakh N
中科院分区：
文献类型：
--
作者：
Boussier J;Munier S;Achouri E;Meyer B;Crescenzo-Chaigne B;Behillil S;Enouf V;Vignuzzi M;van der Werf S;Naffakh N
与大多数RNA病毒一样，流感病毒在复制过程中产生具有大量内部缺失的缺陷病毒基因组（dvg）。越来越多的证据支持这种dvg的生物学相关性。然而，进一步了解dvg产生和生物活性的分子机制取决于检测方法的灵敏度和准确性，即下一代测序（NGS）技术和相关的生物信息学算法。虽然开发了许多算法，但它们的灵敏度和再现性大多是在模拟数据上进行评估的。在这里，我们介绍了DG-seq，一种用于DVG检测和量化的高效管道，以及一套生物对照，用于评估我们的生物信息学算法和上游NGS步骤的性能。使用这些工具，我们首次对两种常用的RNA-seq样品处理方法进行了严格的比较，有或没有PCR预扩增步骤。我们的数据表明，预扩增在灵敏度方面具有有限的优势，并在DVG定量中引入了大小和序列依赖的偏差，从而为支持无预扩增方法提供了强有力的理由。我们进一步研究了野生型和转录缺陷型（PA- k635a或PA- r638a）流感病毒产生dvg的特征，发现与野生型病毒相比，PA突变体产生dvg的多样性和频率更高。最后，我们证明了dvg的显著富集，在缺失断点处显示直接的，富含a / t的序列重复。我们的发现为流感病毒DVG产生的机制提供了新的见解。
Like most RNA viruses, influenza viruses generate defective viral genomes (DVGs) with large internal deletions during replication. There is accumulating evidence supporting a biological relevance of such DVGs. However, further understanding of the molecular mechanisms that underlie the production and biological activity of DVGs is conditioned upon the sensitivity and accuracy of detection methods, that is, next-generation sequencing (NGS) technologies and related bioinformatics algorithms. Although many algorithms were developed, their sensitivity and reproducibility were mostly assessed on simulated data. Here, we introduce DG-seq, a time-efficient pipeline for DVG detection and quantification, and a set of biological controls to assess the performance of not only our bioinformatics algorithm but also the upstream NGS steps. Using these tools, we provide the first rigorous comparison of the two commonly used sample processing methods for RNA-seq, with or without a PCR preamplification step. Our data show that preamplification confers a limited advantage in terms of sensitivity and introduces size- but also sequence-dependent biases in DVG quantification, thereby providing a strong rationale to favor preamplification-free methods. We further examine the features of DVGs produced by wild-type and transcription-defective (PA-K635A or PA-R638A) influenza viruses, and show an increased diversity and frequency of DVGs produced by the PA mutants compared to the wild-type virus. Finally, we demonstrate a significant enrichment in DVGs showing direct, A/T-rich sequence repeats at the deletion breakpoint sites. Our findings provide novel insights into the mechanisms of influenza virus DVG production.
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影响因子：
6.7
作者：
Sun, Yan;Kim, Eun Ji;Lopez, Carolina B.
通讯作者：
Lopez, Carolina B.
影响因子：
14.9
作者：
Routh A;Johnson JE
通讯作者：
Johnson JE
DOI：
10.1261/rna.066910.118
发表时间：
2018-10
期刊：
RNA (New York, N.Y.)
影响因子：
--
作者：
Beauclair G;Mura M;Combredet C;Tangy F;Jouvenet N;Komarova AV
通讯作者：
Komarova AV
影响因子：
30.3
作者：
Poirier EZ;Goic B;Tomé-Poderti L;Frangeul L;Boussier J;Gausson V;Blanc H;Vallet T;Loyd H;Levi LI;Lanciano S;Baron C;Merkling SH;Lambrechts L;Mirouze M;Carpenter S;Vignuzzi M;Saleh MC
通讯作者：
Saleh MC
影响因子：
5.4
作者：
Alnaji, Fadi G.;Holmes, Jessica R.;Brooke, Christopher B.
通讯作者：
Brooke, Christopher B.