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Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing.
Genes (Basel). 2020 08 12; 11(8)G

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.

Authors+Show Affiliations

Legal Medicine Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Torrette, 60126 Ancona, Italy.Virology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Torrette, 60126 Ancona, Italy.Legal Medicine Unit, AOU Ospedali Riuniti, Torrette, 60126 Ancona, Italy.Legal Medicine Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Torrette, 60126 Ancona, Italy.Legal Medicine Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Torrette, 60126 Ancona, Italy.Virology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Torrette, 60126 Ancona, Italy.Virology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Torrette, 60126 Ancona, Italy.Legal Medicine Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Torrette, 60126 Ancona, Italy.Virology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Torrette, 60126 Ancona, Italy.

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

32806776

Citation

Alessandrini, Federica, et al. "Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel By Massive Parallel Sequencing." Genes, vol. 11, no. 8, 2020.
Alessandrini F, Caucci S, Onofri V, et al. Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing. Genes (Basel). 2020;11(8).
Alessandrini, F., Caucci, S., Onofri, V., Melchionda, F., Tagliabracci, A., Bagnarelli, P., Di Sante, L., Turchi, C., & Menzo, S. (2020). Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing. Genes, 11(8). https://doi.org/10.3390/genes11080929
Alessandrini F, et al. Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel By Massive Parallel Sequencing. Genes (Basel). 2020 08 12;11(8) PubMed PMID: 32806776.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing. AU - Alessandrini,Federica, AU - Caucci,Sara, AU - Onofri,Valerio, AU - Melchionda,Filomena, AU - Tagliabracci,Adriano, AU - Bagnarelli,Patrizia, AU - Di Sante,Laura, AU - Turchi,Chiara, AU - Menzo,Stefano, Y1 - 2020/08/12/ PY - 2020/07/01/received PY - 2020/07/30/revised PY - 2020/08/10/accepted PY - 2020/8/19/entrez PY - 2020/8/19/pubmed PY - 2020/8/29/medline KW - COVID-19 KW - SARS-CoV-2 KW - massively parallel sequencing KW - nasopharyngeal swab KW - viral genome JF - Genes JO - Genes (Basel) VL - 11 IS - 8 N2 - 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. SN - 2073-4425 UR - https://www.unboundmedicine.com/medline/citation/32806776/Evaluation_of_the_Ion_AmpliSeq_SARS_CoV_2_Research_Panel_by_Massive_Parallel_Sequencing_ L2 - https://www.mdpi.com/resolver?pii=genes11080929 DB - PRIME DP - Unbound Medicine ER -