S4 flow cell

The libraries were prepared using the Illumina COVIDSeq protocol (Illumina Inc., San Diego, CA, USA). Each previously purified PCR product was processed for tagmentation and adapter ligation using IDT for the Illumina Nextera UD Indexes Set A, B, C and D (384 indexes, 384 samples). Further enrichment and clean-up were performed as per the protocols provided by the manufacturer (Illumina Inc., San Diego, CA, USA). One COVIDSeq-positive control HT (CPC HT) and one non-template control (NTC) were added to the protocol. All libraries were pooled together. The pooled samples were quantified using Qubit 2.0 fluorometer (Invitrogen Inc., MA, USA) and fragment sizes were analysed in Agilent Fragment analyser 5200 (Agilent Inc., Santa Clara, CA, USA). The pooled libraries were further normalised to a 4-nM concentration, and 25 μL of each normalised pool containing index adapter sets were combined in a new microcentrifuge tube to a final concentration of 100 pM and 120 pM. For sequencing, the pooled libraries were denatured and neutralised with 0.2-N NaOH and 400-mM Tris-HCL (pH-8). Replicates of each of the 384 sample pools were loaded onto the S4-flow cell following the NovaSeq-XP workflow, as per the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA). Dual-indexed single-end sequencing with a 36-bp read length was carried out on the NovaSeq 6000 platform.

Cell-free DNA (cfDNA) was extracted from plasma using MagMAX cfDNA isolation kit. After PicoGreen quantification, 47–500 ng of cfDNA were used to make sequencing libraries using the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) with 4 cycles of PCR and pooled equimolar. One sample with sufficient input was prepared PCR-free. Samples were run on a NovaSeq 6000 in a PE150 run, using the NovaSeq 6000 SBS v1 Kit and an S4 flow cell (Illumina). The average coverage per sample was 91X.