Pacbio smrt system

Nucleic acids were extracted from nasopharyngeal swab samples on an MGI SP-960 instrument using the MGIEasy Nucleic Acid Extraction kit and amplified with the Thermofisher TaqPath RT-PCR assay (ThermoFisher, Waltham, MA, USA) on QuantStudio 5 Real-Time PCR systems. Positive results were then classified, according to the TaqPath S gene profile, as: S gene target failure (SGTF), S gene target late detection (SGTL), or non-SGTF/SGTL [21 (link)]. The SGTL profile was defined as a difference of at least 4 Ct between the N and S genes. The Omicron BA.1 variant was identified by its SGTF/SGTL TaqPath profiles (69–70 S gene deletion). Positive specimens with non-SGTF/SGTL profiles (only samples with N Ct ≤ 30) were then tested using the IDTM SARS-CoV-2/VOC Revolution Pentaplex assay (ID solutions, Montpellier, France). This multiplex RT-PCR assay targets the K417N, L452R, and E484K mutations. The Omicron BA.2 variant was identified by the presence of the K417N mutation. Our VOC screening strategy was validated by sequencing a large number of positive nasopharyngeal samples using the Pacific Biosciences (Pacbio) SMRT System [22 (link)].

Genomic DNA was extracted as previously described [41 ]. The genome of Castellaniella defragrans 65Phen was sequenced at the Max Planck Genome Center in Cologne, using the PacBio SMRT system (Pacific Biosciences, Menlo Park, CA). 19858 quality-checked error-corrected reads with at least 4975 bases were de novo assembled to a single contig. The average coverage amounted 91 times and around 4 kb overlapped at each end. Open-reading frames were predicted and annotated by the Rapid Annotations using Subsystems Technology (RAST) pipeline [42 (link)]. The G+C content was calculated using Artemis [43 (link)]. Putative prokaryotic promoters were predicted with BPROM (http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) and putative terminators were identified using WebGeSTer [44 (link)]. ANI was calculated according to Goris et al.[45 (link)]. The complete genome sequence of Castellaniella defragrans 65Phen has been deposited at GenBank under the accession number HG916765.