Next Article in Journal
Insights into Mobile Genetic Elements of the Biocide-Degrading Bacterium Pseudomonas nitroreducens HBP-1
Next Article in Special Issue
Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation during a Pandemic
Previous Article in Journal
Karyotypic Evolution of Sauropsid Vertebrates Illuminated by Optical and Physical Mapping of the Painted Turtle and Slider Turtle Genomes
Previous Article in Special Issue
Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler
Open AccessArticle

Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing

1
Legal Medicine Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Torrette, 60126 Ancona, Italy
2
Virology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Torrette, 60126 Ancona, Italy
3
Legal Medicine Unit, AOU Ospedali Riuniti, Torrette, 60126 Ancona, Italy
*
Author to whom correspondence should be addressed.
Genes 2020, 11(8), 929; https://doi.org/10.3390/genes11080929
Received: 1 July 2020 / Revised: 30 July 2020 / Accepted: 10 August 2020 / Published: 12 August 2020
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. View Full-Text
Keywords: SARS-CoV-2; viral genome; massively parallel sequencing; nasopharyngeal swab; COVID-19 SARS-CoV-2; viral genome; massively parallel sequencing; nasopharyngeal swab; COVID-19
Show Figures

Figure 1

MDPI and ACS Style

Alessandrini, F.; Caucci, S.; Onofri, V.; Melchionda, F.; Tagliabracci, A.; Bagnarelli, P.; Di Sante, L.; Turchi, C.; Menzo, S. Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing. Genes 2020, 11, 929.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Search more from Scilit
 
Search
Back to TopTop