SARS-CoV-2 N Gene Sequencing Protocol

Sequencing libraries were prepared from unpurified RT-PCR products using the NEBNext Ultra DNA Library Preparation Kit (New England Biolabs Inc., Ipswich, MA) modified for very short insert sizes [6 (link)]. The libraries were pooled at approximately equimolar ratios and sequenced using the Illumina MiSeq Micro v2 kit (300 cycles) (Illumina Inc., San Diego, CA). Demultiplexed FastQ files were generated using bcl2fastq v2.20 with default settings but adding a setting to preserve short-trimmed reads. Adapters and low-quality bases were trimmed and merged using FastP v0.20.1 with default parameters but again removing the limit for minimum read length [7 (link)]. Merged reads were mapped to the SARS-CoV2 Wuhan-Hu-1 (NCBI: MN908947) N gene target sequences in Geneious Prime 2019 using high sensitivity settings, modified to include a minimum mapping quality of 20 and a maximum gap size of 2 bp [8 ]. Unmapped reads were then mapped to all nine N1, N2, and N3 primer and probe sequences using the same parameters. All alignments were visually inspected to verify accuracy.