Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing
Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing
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DOI：
10.3390/genes11080929
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发表时间：
2020-08-01
期刊：
影响因子：
3.5
通讯作者：
Menzo, Stefano
中科院分区：
文献类型：
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作者：
Alessandrini, Federica;Caucci, Sara;Menzo, Stefano
深入了解SARS-CoV-2的遗传特征，对于在不同地理区域追踪正在发生的大流行，以及设计和制定早期诊断程序、治疗策略、公共卫生干预措施和疫苗至关重要。我们描述了Ion AmpliSeq (TM) SARS-CoV-2研究小组通过大规模平行测序（MPS）测定的方案和初步结果。该面板允许通过重叠扩增子进行靶向测序，从而提供特定，准确和高通量的分析。为了促进病毒RNA特异性逆转录，评估了一种改良的逆转录反应，该反应包括使用来自Ion AmpliSeq SARS-CoV-2研究小组的SARS-CoV-2特异性引物池。本研究的目的是评估Ion AmpliSeq (TM) SARS-CoV-2研究小组在不同样本中全病毒基因组测序中的有效性。从不同患者的10株病毒分离株和1份鼻咽拭子中获得SARS-CoV-2序列数据。用12个PCR循环扩增的10个分离样品的平均深度值高于用20个PCR循环扩增的2个分离样品。使用靶向特异性逆转录处理的鼻咽拭子也获得了较高的平均深度值。相对覆盖深度（relative depth of coverage, rDoC）分析表明，当使用12个PCR循环时，所有的目标区域都被扩增，测序覆盖率高，而在扩增20个循环时，扩增的均匀性较差，许多目标区域没有或覆盖率低。本研究结果表明，Ion AmpliSeq SARS-CoV-2研究小组可以通过12个PCR循环进行文库扩增，从分离物中获得10 ng无dna的病毒RNA和从鼻咽拭子中获得1 ng无dna的病毒RNA，实现快速、高通量的SARS-CoV-2全基因组测序。与根据制造商说明设置的逆转录反应相比，改进的RT-PCR方案在鼻咽拭子上产生了更好的结果。
Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq (TM) SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq (TM) SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.