Abstract
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™ 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™ 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.
Highlights
SARS-CoV-2, first isolated in Wuhan, China, in December 2019 [1] is a new coronavirus responsible for the severe pulmonary/systemic disease called COVID-19
The library prepared from the nasopharyngeal swab, reverse transcribed with a modified protocol by using the SARS-CoV-2 specific primer pools from the Ion AmpliSeq SARS-CoV-2 Research Panel, displayed the best amplicon curve distribution and was very similar to the expected pattern (Figure 1b)
We describe the protocols and the first results of a targeted massively parallel sequencing (MPS) assay by Ion both samples displayed a G4255T substitution in 100% of GeneStudioTM S5 System
Summary
SARS-CoV-2, first isolated in Wuhan, China, in December 2019 [1] is a new coronavirus responsible for the severe pulmonary/systemic disease called COVID-19. Genes 2020, 11, 929 spread in Southeast Asia and subsequently in Europe and the rest of the world. In Italy, the first two cases were Chinese tourists diagnosed on 29 January 2020 [2], while the first documented case of local transmission, in Lombardy, was on 22 February 2020. Several clusters were discovered in northern regions (Lombardy, Emilia-Romagna, and Veneto), followed by rapid spread of the epidemic towards central regions such as Marche. The first COVID-19 patient was diagnosed by the Virology Laboratory of Ancona University Hospital on the 25 February 2020. Following the spread of the infection in Europe and all over the globe, the World Health
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