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.
The precision of the ID Identity Panel kit was assessed on a large set of challenging forensic samples. A threshold of 50 reads for locus call reduces the frequency of sequencing errors. Replicate analyses assure a low/null rate of typing errors. The high number of markers of the kit assures a random match of probability ≤ 1.6 x 10-13 even for the most challenging samples. PCR-MPS of SNP markers is the ideal approach to the analysis of LCN and degraded DNAs.
The study of microbiomes has enormous potential for forensic science because microorganisms are ubiquitous and particular communities of microbes are often associated with specific processes or environments. With recent advances in microbiome science, new opportunities exist for microbiome technologies in forensic science (PMI estimation, location of clandestine graves, soil analysis and personal identification). Before a new technology is accepted by the forensic science, it requires an initial validation phase.The aim of our study was to evaluate if the DNA IQ™ Casework Pro Kit for Maxwell® 16 (Promega) is suitable for microbial DNA extraction, without modifications. Ten bacterial strains were selected and subjected to the GenElute Bacterial Genomic DNA extraction protocol (Sigma-Aldrich) and to the DNA IQ™ Casework Pro Kit for Maxwell® 16 protocol. Extracted DNA was quantified and submitted to NGS analysis on an Ion S5 NGS System. Data were analyzed using the Ion Reporter Software metagenomics workflow. Our work has shown that it is possible to purify both microbial and human DNA using the Promega kit, thus making it possible to analyze both human and microbial DNA from a single trace, a pivotal factor in forensics where the quantities of biological material available are usually very limited.
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