Agilent high sensitivity d1000 screentape assay

DNA concentration was measured using the Qubit Fluorometer and the Qubit dsDNA BR Assay Kit (ThermoFisher; Q32853). 250 ng input DNA was used in the sequencing library preparation. Indexed sequencing libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina; 20018705) and IDT^® for Illumina Nextera™ DNA CD Indexes (Illumina; 20018708). 5 cycles of PCR were used. Libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (ThermoFisher Scientific; Q32854). Library fragment-length distribution was assessed using the Agilent TapeStation 4,200 and the Agilent High Sensitivity D1000 ScreenTape Assay (Agilent; 5067-5584 and 5067-5585). Final library quantification was performed using the KAPA Library Quantification Kit for Illumina (Roche; KK4824) and the library was sequenced on an Illumina MiSeq using the MiSeq Reagent Kit v3 (600 cycle) (Illumina; MS-102-3,003) to generate 300-bp paired-end reads.

Total RNA was extracted with NucleoSpin RNA Plus XS (Macherey–Nagel GmbH & Co) from the 9 samples following the manufacturer’s instructions. Total RNA sample integrity and concentration was measured by Agilent High Sensitivity RNA ScreenTape Assay (Agilent 2200 TapeStation system, Agilent Technologies, Inc., Santa Clara, US). Sample concentrations for quantification were determined by Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc., Waltham, USA). During extraction, genomic DNA was removed by gDNase during on-column DNA digestion. The purified RNA was dissolved in 20 µL RNase-free water and stored at − 80 °C. The cDNA was generated and amplified in 9 cycles with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio, Inc. Mountain View, USA). Libraries were generated (Nextera XT DNA Library Prep Kit, Illumina, San Diego, USA) and sample quality was checked by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, USA) and by Agilent High Sensitivity D1000 ScreenTape Assay (Agilent 2200 TapeStation system). Custom index primers were tagged to the libraries to allow for multiplexing during RNA-sequencing. Libraries were quantified, normalised based on measurements determined by Qubit 2.0 Fluorometer, and sequenced on the Illumina NextSeq 500 system (150 cycles). The raw RNA-Seq data was deposited and released with the BioProject accession number of PRJNA635095.

DNA concentrations were measured using the Qubit Fluorometer and the Qubit dsDNA BR Assay Kit (ThermoFisher Scientific, Loughborough, UK) and 250 ng were used for sequencing library preparation. Indexed sequencing libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA) and Nextera DNA CD Indexes (Illumina, San Diego, CA, USA). Libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (ThermoFisher Scientific, Loughborough, UK). Library fragment-length distributions were assessed using the Agilent TapeStation 4200 and the Agilent High Sensitivity D1000 ScreenTape Assay (Agilent, Santa Clara, CA, USA). Libraries were pooled in equimolar amounts and final library quantification performed using the KAPA Library Quantification Kit for Illumina (Roche, Basel, Switzerland). The library pool was sequenced on an Illumina MiSeq using the MiSeq Reagent Kit v2 (500 cycle) (Illumina, San Diego, CA, USA) to generate 250 bp paired-end reads. The whole genome shotgun was deposited in GenBank as a BioProject under Accession PRJNA826427.

Viral enrichment of clinical extracts, in vitro culture isolates, and the synthetic SARS-CoV-2 positive control spiked into negative culture supernatant was performed using the Illumina RNA Prep and Enrichment with the Respiratory Viral Oligo Panel (RVOP) v2 (Illumina, United States). This probe-based capture technique was selected as it was designed to generate near full length SARS-CoV-2 genomic sequences with even coverage in mutagenic regions. RNA denaturation, first and second strand cDNA synthesis, cDNA tagmentation, library clean-up and normalisation were performed according to the manufacturer’s instructions. Individual libraries were pooled in 3-plex reactions for probe hybridisation based on each samples SARS-CoV-2 viral load. The final probe hybridisation step was held overnight at 58°C. The enriched library was purified, and the concentration and fragment size were quantified using the Qubit™ 1x dsDNA High Sensitivity Assay (Thermo Fisher Scientific, United States), and Agilent High Sensitivity D1000 ScreenTape assay on the Agilent 4200 Tapestation (Agilent, Germany), respectively. The libraries were sequenced using 2 × 74 bp runs on the Illumina MiniSeq™ or iSeq (Illumina, United States) and multiplexed with the aim of producing 2 × 10⁶ raw reads per library.