SARS-CoV-2 complete genome sequencing from the Italian Campania region using a highly automated next generation sequencing system

Rachiglio, Anna Maria; Sabato, Luca De; Roma, Cristin; Cennamo, Michele; Fiorenza, Mariano; Terracciano, Daniela; Pasquale, Raffaella; Bergantino, Francesca; Cavalcanti, Ernesta; Botti, Gerardo; Vaccari, Gabriele; Portella, Giuseppe; Normanno, Nicola

doi:10.1186/s12967-021-02912-4

Cited by 15 publications

(11 citation statements)

References 22 publications

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“…The available literature shows the full-genome sequences can be obtained even from samples with less than 100 viral RNA copy number per reaction by analogous workflows (Ion AmpliSeq SARS-CoV-2 Research Panel and Ion Torrent Genexus Integrated System) [28]. This is just below the lowest number of viral RNA copies for which we have obtained high-quality results (156 viral RNA copies per reaction, sample 365; see Table S5).…”

Section: Discussionmentioning

confidence: 75%

“…Consistent with our study are findings by Jacot et al [29] who suggest C T ≥ 30 (78 or less of viral RNA copy number per reaction) can serve as an initial sequencing success predictor. An analysis comparing the Ion AmpliSeq Panel and Illumina-MiSeq ARCTIC Protocol showed both methods are overall equally effective, with the automation of all steps of library preparation within the TFS solution considered a strong advantage [30], which was expressed also by Rachiglio et al [28]. In the beforementioned study [30], one low-quality sample (C T 32,5) achieved 89% coverage with MiSeq-based ARCTIC pipeline, while it failed with the SARS-CoV-2 AmpliSeq Research panel, suggesting the TFS solution may be inferior to the MiSeq-based one in cases of low-template material.…”

Section: Discussionmentioning

confidence: 92%

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SARS-CoV-2 Whole-Genome Sequencing by Ion S5 Technology—Challenges, Protocol Optimization and Success Rates for Different Strains

Szargut

Cytacka

Serwin

et al. 2022

Viruses

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The COVID-19 pandemic demonstrated how rapidly various molecular methods can be adapted for a Public Health Emergency. Whether a need arises for whole-genome studies (next-generation sequencing), fast and high-throughput diagnostics (reverse-transcription real-time PCR) or global immunization (construction of mRNA or viral vector vaccines), the scientific community has been able to answer all these calls. In this study, we aimed at the assessment of effectiveness of the commercially available solution for full-genome SARS-CoV-2 sequencing (AmpliSeq™ SARS-CoV-2 Research Panel and Ion AmpliSeq™ Library Kit Plus, Thermo Fisher Scientific). The study is based on 634 samples obtained from patients from Poland, with varying viral load, assigned to a number of lineages. Here, we also present the results of protocol modifications implemented to obtain high-quality genomic data. We found that a modified library preparation protocol required less viral RNA input in order to obtain the optimal library quantity. Concurrently, neither concentration of cDNA nor reamplification of libraries from low-template samples improved the results of sequencing. On the basis of the amplicon success rates, we propose one amplicon to be redesigned, namely, the r1_1.15.1421280, for which less than 50 reads were produced by 44% of samples. Additionally, we found several mutations within different SARS-CoV-2 lineages that cause the neighboring amplicons to underperform. Therefore, due to constant SARS-CoV-2 evolution, we support the idea of conducting ongoing sequence-based surveillance studies to continuously validate commercially available RT-PCR and whole-genome sequencing solutions.

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Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 92%

SARS-CoV-2 Whole-Genome Sequencing by Ion S5 Technology—Challenges, Protocol Optimization and Success Rates for Different Strains

Szargut

Cytacka

Serwin

et al. 2022

Viruses

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“…This report includes a review of the first cases profiled using a novel highly automated gene sequencing system [13]. The automated nature of this system enables its use directly within a diagnostic histopathology laboratory, eliminating transfers of specimens and reports.…”

Section: Introductionmentioning

confidence: 99%

Point of Care Molecular Testing: Community-Based Rapid Next-Generation Sequencing to Support Cancer Care

et al. 2022

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Biomarker data are critical to the delivery of precision cancer care. The average turnaround of next-generation sequencing (NGS) reports is over 2 weeks, and in-house availability is typically limited to academic centers. Lengthy turnaround times for biomarkers can adversely affect outcomes. Traditional workflows involve moving specimens through multiple facilities. This study evaluates the feasibility of rapid comprehensive NGS using the Genexus integrated sequencer and a novel streamlined workflow in a community setting. Methods: A retrospective chart review was performed to assess the early experience and performance characteristics of a novel approach to biomarker testing at a large community center. This approach to NGS included an automated workflow utilizing the Genexus integrated sequencer, validated for clinical use. NGS testing was further integrated within a routine immunohistochemistry (IHC) service, utilizing histotechnologists to perform technical aspects of NGS, with results reported directly by anatomic pathologists. Results: Between October 2020 and October 2021, 578 solid tumor samples underwent genomic profiling. Median turnaround time for biomarker results was 3 business days (IQR: 2–5). Four hundred eighty-one (83%) of the cases were resulted in fewer than 5 business days, and 66 (11%) of the cases were resulted simultaneously with diagnosis. Tumor types included lung cancer (310), melanoma (97), and colorectal carcinoma (68), among others. NGS testing detected key driver alterations at expected prevalence rates: lung EGFR (16%), ALK (3%), RET (1%), melanoma BRAF (43%), colorectal RAS/RAF (67%), among others. Conclusion: This is the first study demonstrating clinical implementation of rapid NGS. This supports the feasibility of automated comprehensive NGS performed and interpreted in parallel with diagnostic histopathology and immunohistochemistry. This novel approach to biomarker testing offers considerable advantages to clinical cancer care.

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“…Plitnick et al, 2021, directly compared the performance of the SARS-CoV-2 AmpliSeq Research Panel to the results obtained with the Illumina MiSeqbased ARTIC Next ow analysis pipeline (30). Post-bioinformatic analysis of data from such studies showed that both methods produced similar levels of coverage (> 98%) across a broad range of viral loads (Ct values of 15.56 to 32.54 [median, 22.18]) and that both approaches sequenced SARS-CoV-2 effectively (30,32). Although the bioinformatic analysis pipelines used in this study differ, the ndings of our study are similar to those documented in the above study by Plitnick et al, 2021.…”

Section: Discussionmentioning

confidence: 99%

Comparing SARS-CoV-2 sequencing methodologies during early phase detection of the Delta variant in South Africa

Ramphal

Tshiabuila

Ramphal

et al. 2023

Preprint

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Background: Genomic surveillance, with the aid of next-generation sequencing (NGS) technologies, revolutionized the SARS-CoV-2 pandemic. Coupled with high-performance analysis software, methodologies such as the Ion Torrent S5 and Illumina MiSeq dramatically improved the genomic surveillance capacity within South Africa during the height of the pandemic. Using de-identified remnant samples collected from Eastern Cape and analysis software, Genome Detective and NextClade, we compared the sequencing process, genomic coverage, quantification of mutations, and clade classification from sequence data generated by these two common “benchtop” NGS platforms. Results: Sequence data analysis revealed success rates of 175/183 (96%) and 172/183 (94%) on the Ion Torrent S5 and Illumina MiSeq, respectively. Internal quality metrics were assessed in terms of genomic coverage (>80%) and the number of mutations identified (<100). A greater number of higher-genomic coverage sequences were generated on the Ion Torrent S5 (99%) than on the Illumina MiSeq (80%) and <100 mutations was obtained by both platforms. Ion Torrent S5 generated high coverage sequences from samples having a broader range of viral loads (VL) compared to the Illumina MiSeq, which was less successful in sequencing samples with lower viral loads. Clade assignments were comparable across platforms which accurately differentiated between Beta (<45%) and Delta (≤30%) VOCs. A disparity in clade assignment was observed in <10% of sequences due to poor coverage obtained on the Illumina MiSeq, followed by a failure rate of ≤6% across the two platforms. Manual library preparation found both methods similar in terms of sample processing, handling of larger sample quantities, and clade assignment for SARS-CoV-2. Variability between the Ion Torrent S5 and Illumina MiSeq was observed in sequencing run duration (3,5 hrs vs 36 hrs), sequencing process (semi-automation vs manual), genomic coverage (99% vs 80%), and viral load requirements (broad range vs high VL). Conclusion: The Illumina MiSeq and Ion Torrent S5 are both reliable platforms capable of performing WGS with the use of amplicons and providing specific, accurate, and high throughput analysis of the SARS-CoV-2 whole viral genomes. Both sequencing platforms are feasible platforms for the genomic surveillance of SARS-CoV-2, each with its specific advantages and trade-offs.

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SARS-CoV-2 complete genome sequencing from the Italian Campania region using a highly automated next generation sequencing system

Cited by 15 publications

References 22 publications

SARS-CoV-2 Whole-Genome Sequencing by Ion S5 Technology—Challenges, Protocol Optimization and Success Rates for Different Strains

SARS-CoV-2 Whole-Genome Sequencing by Ion S5 Technology—Challenges, Protocol Optimization and Success Rates for Different Strains

Point of Care Molecular Testing: Community-Based Rapid Next-Generation Sequencing to Support Cancer Care

Comparing SARS-CoV-2 sequencing methodologies during early phase detection of the Delta variant in South Africa

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