Barcoded primers

Purified biotinylated transcripts were depleted of mature ribosomal RNAs using a NEBNext® rRNA Depletion Kit (New England Biolabs, cat# E6350), and used to prepare stranded sequencing libraries with a NEBNext Ultra II Directional RNA library Prep kit for Illumina and barcoded primers (New England Biolabs, cat# E7765) according to the manufacturer’s instruction. The barcoded libraries were quantified using NGSBIO Library Quant Kit Blue for Illumina® (PCR Biosystems, cat# PB71.15-01) and pooled prior to sequencing. Single-read sequencing was performed at the Oxford Genomics Centre, UK using a NextSeq 500 platform (Illumina, NextSeq 500/550 v2.5 Kits, 75 cycles) at ∼20 million reads per demultiplexed sample per single lane of the NextSeq 500 flow cell.

To validate COVseq, we generated individual libraries from 29 left-over samples (OAS-29 samples in Supplementary Data 4) using the NEBNext Ultra II FS DNA Library Prep Kit (NEB, cat. no. E7805L) following the manufacturer’s instructions. Briefly, we used 250 ng of the pooled purified amplicons from each sample as input to prepare a library. First, we enzymatically fragmented the amplicons for 7 min at 37 °C followed by incubation for 30 min at 65 °C to achieve a target size around 200 bp. After fragmentation, we performed end-repair and adapter ligation in the same tube followed by purification of the fragments using a 0.8 vol/vol ratio of Ampure XP beads, following the manufacturer’s instructions. We amplified adapter-ligated DNA fragments by three PCR cycles with barcoded primers (NEB, cat. no. E7500S) following the manufacturer’s instructions and purified the PCR product with a 0.9 vol/vol ratio of Ampure XP beads. We assessed the size distribution and concentration of the libraries on a Bioanalyzer 2100 (Agilent Technologies, cat. no. G2943CA) using the High Sensitivity DNA kit (Agilent Technologies, cat. no. 5067–4626).