Qiasymphony dsp mini kit 200

PEG samples were extracted using either the Qiagen RNA viral extraction kit or the Qiasymphony-DSP mini kit 200 (Qiagen, UK) with offboard lysis. RNA was tested for SARS-CoV-2 using the CDC N1 assay (IDT) and qScript 1-step master mix (10ul master mix, 1.5 primer/probe (FAM), 3.5ul water, and 5ul RNA) with a positive and negative control. We also ran a standard curve using a serial dilution of a SARS-CoV-2 genomic fragment from 1,000,000 to 10 copies/ul.

Samples were extracted using the Qiasymphony-DSP mini kit 200 (Qiagen, UK) with offboard lysis or manually using the Qiagen mini viral extraction kit. Samples were then tested using the CDC N1 assay to confirm the Ct values before sequencing. ARTIC protocol V2 sequencing protocol was used until June 2021, after which we switched to the V3 protocol. ARTIC version 3 primers were used for the tiling PCR until we switched to the University of Zambia (UNZA) primer set that provided better results for Delta VOC in August 2021 (data not shown) [20 (link)]. Initially two primer pools were used, however a third pool was made for primer pairs that commonly had lower depth compared to the average (details Additional file 1: Table S1). PCR cycling conditions were adapted to the new sequencing primers, with annealing temperature changed to 60 °C. Sequencing was carried out with the Oxford Nanopore Technologies MinION sequencer. Samples that had poor coverage (< 70%) with the ARTIC primer set were repeated with the UNZA primer set.

Samples were extracted using the Qiasymphony-DSP mini kit 200 (Qiagen, UK) with offboard lysis. Samples were then tested using the CDC N1 assay to confirm the Ct values before sequencing. Samples with a Ct value <27 were sequenced. The following sequencing protocols were used; ARTICv2 and v3 was used from November 2020 from July 2021 to July 2021 [16 ] and UNZA [17 (link)] from August 2021 on wards. Initially two primer pools were used, however a third pool was made for primer pairs that commonly had lower depth compared to the average [15 (link)]. PCR cycling conditions were adapted to the new sequencing primers, with annealing temperature changed to 60°C. Sequencing was carried out with the Oxford Nanopore Technologies MinION sequencer. Samples that had poor coverage (<70%) with the ARTIC primer set were repeated with the UNZA primer set.
For analysis of sequencing data, the lineage of each consensus genome was identified using pangolin with the following versions; pangolin v3.1.17, pangolearn 2021-12-06, constellations v0.1.1, scorpio v0.3.16, pango-designation used by pangoLEARN/Usher v1.2.105, pango-designation aliases v1.2.122 [18 (link)]. Samples were re-analysed when the Pangolin database was updated. The run was repeated if there was contamination in the negative control.