title: InCOV0139
design: Highly multiplexed PCR amplicon NGS was performed on the D16nasopharyngeal swab, using the ARTIC V3 amplicon primer scheme, 7 and modifiedfrom the ARTIC protocol, 8 to approach the low copy number sample, as follows. RNAwas extracted from nasopharyngeal swab samples using the miRNeasy kit (Qiagen,Germantown, MD) and cDNA was synthesized with the LunaScript RT SuperMix kit(NEB, Ipswich, MA). The set of 98 SARS-CoV-2-specific primer pairs were synthesized 7and pooled for multiplex PCR using the following PCR conditions: 98 o C for 30 seconds,followed by 40 cycles of 95 o C for 15 seconds, 65 o C for 5 minutes. The primer setgenerated overlapping PCR fragments covering the entire viral genome, the averageamplicon length being 400 bp with an approximate 100 bp overlap. After cleaning up thePCR products with AMPure XP beads (Beckman Coulter, Indianapolis, IN),Fragmentase (NEB, Ipswich, MA) was added to break down the PCR products to about200 bp for library construction. The sequencing libraries were prepared with NEBNextUltra II kit for Illumina (NEB, Ipswich, MA) following manufacturers protocol and eachlibrary was assigned with a unique 6-base index. Libraries ranging in size from 300 to 500 bp were selected with Pippin HT (Sage Science, Beverly, MA) and the size wasconfirmed with the Bioanalyzer (Agilent, Santa Clara, CA). Libraries were quantified withthe KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems,Wilmington, MA) and pooled at a final concentration of 5nM. Pooled libraries were runon NextSeq (Illumina, San Diego, CA) using 300 cycles paired end reagent kit. The rawsequence data was demultiplexed using qcat (https://github.com/nanoporetech/qcat)and assembled against reference genome (NC_045512.2) with Sequencher (GeneCodes, Ann Arbor, MI), allowing for no more than 2 mismatches within a 40 base pairwindow. Ambiguous bases were amplified via PCR and sent for Sanger sequencing.The mutation at C18877T is confirmed via Sanger sequencing (Primers: ForwardCGCACCTGTTGTCTATGTGA, Reverse: GAACCTTTCTACAAGCCGCAT,Sequencing: GGTTTTACAGGTAACCTACA), but amplification was unsuccessful atother positions.