Nextera dna flex prep kit

The ARTIC SARS‐CoV‐2 protocol (https://artic.network/ncov-2019) was used for amplicon generation. Sequencing was performed using the methods outlined.¹³, ¹⁴ Briefly, nucleic acid extraction was performed¹⁵ and cDNA was generated by the ARTIC protocol. After cDNA synthesis, two multiplex PCR tiling reactions were prepared using the ARTIC V3, V4, and V4.1 primers (depending on sampling date). cDNA was combined with Q5 High‐Fidelity 2X Master Mix (NEB, USA). To mix #1, 10 μM of ARTIC primer pool #1 were added and 10 μM of ARTIC primer pool #2 were added to mix #2. Cycling was then performed as follows: 98°C for 30 s followed by 35 cycles of 98°C for 15 s and 65°C for 5 min. Following this reaction, products were combined, and a clean‐up with AMPure XP beads was performed (Beckman Coulter, USA). The quantity of amplicons was measured with the Qubit 4.0 fluorometer using the 1X dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). The sequencing libraries were prepared using the Nextera DNA Flex Prep kit (Illumina, USA) as per manufacturer's instructions. Paired‐end (2 × 150 bp) sequencing was performed on a MiniSeq with a 300‐cycle reagent kit (Illumina, USA).

The appropriate volume of each pool of amplicons was adjusted to have a total quantity of 100 ng of amplicon input (50 ng of each) per sample. The sequencing-ready libraries were prepared using the Nextera DNA Flex Prep kit (Illumina, United States). The libraries were qualified on an Agilent Technologies 2100 Bioanalyzer using a high-sensitivity DNA chip following the manufacturer’s instructions and quantified with the Qubit 2.0 fluorometer using the dsDNA HS Assay Kit (Thermo Fisher Scientific, United States). The resulting libraries were sequenced using the iSeq100^TM system (Illumina, United States) and in two multiplexed runs were performed generating 2 × 151 bp read length data during a 17 h run time (Figure 1).