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.

Background: Infection related to Coronavirus-19 (CoV-2) is pandemic affecting more than 4 million people in 187 countries worldwide. By May 10, 2020, it caused more than 280 000 deaths all over the world. Preliminary data reported a high prevalence of CoV-2 infection and mortality due to severe acute respiratory syndrome related CoV-2 (SARS-CoV-2) in kidney-transplanted patients (KTRs). Nevertheless, the outcomes and the best treatments for SARS-CoV-2-affected KTRs remain unclear.

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Five distinct cryptic lineages (clades I-V) have recently been recognized in the Escherichia genus. The five clades encompass strains that are phenotypically and taxonomically indistinguishable from Escherichia coli sensu stricto; however, scant data are available on their ecology, virulence and pathogenic properties. In this study 20 cryptic E. coli strains isolated from marine sediments were investigated to gain insights into their virulence characteristics and genetic traits. The ability to adhere to intestinal cells was highest among clade V strains, which also harbored the genes involved in gut colonization as well as the genes (pduC and eut operon) typically found in environmentally adapted E. coli strains. The pduC gene was significantly associated with clade V. Multilocus sequence typing of three representative clade V isolates revealed new sequence types (STs) and showed that the strains shared two allelic loci (adk 51 and recA 37). Our findings suggest that cryptic Escherichia lineages are common in coastal marine sediments and that this habitat may be suitable for their growth and persistence outside the host. On the other hand, detection in clade V strains of a gene repertoire and adhesion properties similar to those of intestinal pathogenic strains could indicate their potential virulence. It could be argued that there is a dual nature of cryptic clade V strains, where the ability to survive and persist in a secondary habitat does not involve the loss of the host-associated lifestyle. Clade V could be a group of closely related, environmentally adapted E. coli strains.