Covidseq test kit

Viral RNA was extracted from 180 μl of each saliva sample using the QIAamp 96 Viral RNA Kit with the QIAcube HT (Qiagen) using the following settings with a filter plate: the lysed sample was premixed eight times before subjecting to vacuum for 5 min at 25 kP and vacuum for 3 min at 70 kPa. Following three washes using the same vacuum conditions above, the samples were eluted in 100 μl AVE buffer followed by a final vacuum for 6 min at 60 kPa. Nine microliters of RNA was used for cDNA synthesis and library preparation using the COVIDSeq Test kit (Illumina) and Mosquito HV Genomics Liquid Handler (SPT Labtech Inc.). The size and purity of the library were determined using the 4200 TapeStation System (Agilent) and the Qubit dsDNA HS Assay Kit (Life Technologies) according to the manufacturer's instructions. Constructed libraries were pooled and sequenced using the NovaSeq. 6000 Sequencing System SP Reagent Kit and the NovaSeq Xp 2‐Lane Kit. Illumina's DRAGEN pipeline was used to derive sample consensus sequences, which were filtered based on a minimum of 70% coverage of the genome.

Clinical samples were received in The Jackson Laboratory Clinical Genomics Laboratory (CGL) as part of a statewide (Connecticut) COVID-19 surveillance program, with the majority of samples representing asymptomatic screening of nursing home and assisted living facility residents and staff. Total nucleic acids were extracted from anterior nares swabs in viral transport media or saline (200μL) using the MagMAX Viral RNA Isolation kit (ThermoFisher) on a KingFisher Flex purification system. Samples were tested for the presence of SARS-CoV-2 RNA using the TaqPath COVID-19 Combo Kit (ThermoFisher). For this analysis, only the CT values from the N gene primer/probe set were used (Figure 4).
Samples with CT values ≤30 for the N gene target were prepared for sequencing using the Illumina COVIDSeq Test kit. Sequencing was performed on an Illumina NovaSeq or NextSeq in the CGL. Data analysis was performed using the DRAGEN COVID Lineage App in BaseSpace Sequence Hub. Sequences with >80% of bases with non-N basecalls and ≥1500-fold median coverage were considered successful and were submitted to GISAID. Lineages were assigned using pangolin v.2.4.2 (O’Toole et al., 2021 ) and the most current version of the pangoLEARN assignment algorithm.

Clinical samples were received in The Jackson Laboratory Clinical Genomics Laboratory (CGL) as part of a statewide COVID-19 surveillance program, with the majority of samples representing asymptomatic screening of nursing home and assisted living facility residents and staff. Total nucleic acids were extracted from anterior nares swabs in viral transport media or saline (200μL) using the MagMAX Viral RNA Isolation kit (ThermoFisher) on a KingFisher Flex purification system. Samples were tested for the presence of SARS-CoV-2 RNA using the TaqPath COVID-19 Combo Kit (ThermoFisher). Samples with cycle thresholds ≤30 for the N gene target were prepared for sequencing using the Illumina COVIDSeq Test kit. Sequencing was performed on an Illumina NovaSeq or NextSeq in the CGL. Data analysis was performed using the DRAGEN COVID Lineage App in BaseSpace Sequence Hub. Sequences with >80% of bases with non-N basecalls and ≥1500-fold median coverage were considered successful and were submitted to GISAID. Lineages were assigned using pangolin v.2.4.2²⁹ and the most current version of the pangoLEARN assignment algorithm.