V3 miseq flowcell

Amplicons were generated by a SARS-CoV-2 specific multiplex PCR^{46 (link)} (for primer sequences, see Table S3). Amplicons were purified with 0.8x AMPure XP beads (Beckman Coulter) and 100 ng of DNA was converted into paired-end Illumina sequencing libraries using the KAPA HyperPlus library preparation kit (Roche), following the manufacturer’s recommendations, to enable subsequent sequencing of multiple libraries in a single Illumina V3 MiSeq flowcell (2 × 300 cycles). Multiplex Adaptors (KAPA Unique Dual-Indexed Adapters Kit (Roche)) with indexes were used. FASTQ files were then imported to the CLC Genomics Workbench v20.0.3 (QIAGEN) for analysis. First, sequences were trimmed off 33 base pairs on both the 3′ and 5′ ends to remove primer sequences and also using Phred quality score threshold of 20. The trimmed sequences were mapped to the reference sequence (GISAID ID EPI_ISL 406862) with the following default parameters (match score = 1, mismatch cost = 2, insertion cost = 3, length fraction = 0.5, and similarity fraction = 8). Variants were called with the Basic Variant Detection tool. Single nucleotide polymorphisms that were present in both the forward and reverse reads with a 100x minimum coverage and a minimum variant count of 5 (5%) were called.

Amplicons were generated by a SARS-CoV-2 specific multiplex PCR as described above for the whole genome sequencing. Amplicons were purified with 0.8x AMPure XP beads (Beckman Coulter) and 100 ng of DNA was converted into paired-end Illumina sequencing libraries using KAPA HyperPlus library preparation kit (Roche), following the manufacturer’s recommendations, to enable subsequent sequencing of multiple libraries in a single Illumina V3 MiSeq flowcell (2×300 cycles). Multiplex Adaptors (KAPA Unique Dual-Indexed Adapters Kit (Roche)) with indexes were used. FASTQ files were then imported to the CLC Genomics Workbench v20.0.3 (QIAGEN) for analysis. First, sequences were trimmed off 33 base pairs on both the 3′ and 5′ ends to remove primer sequences and also using Phred quality score threshold of 20. The trimmed sequences were mapped to the reference sequence (GISAID ID EPI_ISL 406862) with the following default parameters (match score = 1, mismatch cost = 2, insertion cost = 3, length fraction = 0.5 and similarity fraction = 8). Variants were called with the Basic Variant Detection tool. Single nucleotide polymorphisms that were present in both the forward and reverse reads with a 200x minimum coverage and a minimum variant count of 10 (5%) were called.