High output kit v2.5 300 cycles

We sequenced all the NEBNext libraries on the NextSeq 500 system from Illumina using the High Output Kit v2.5 (75 Cycles) (Illumina, Cat. No. 20024906). For COVseq libraries, we used the following kits and platforms: (i) Supernatant sample: High Output Kit v2.5 150 Cycles (Illumina, Cat. No. 20024907) on NextSeq; (ii) OAS-29 samples: High Output Kit v2.5 300 Cycles (Illumina, Cat. No. 20024908) on NextSeq and Reagent Kit v3 600 Cycles (Illumina, Cat. No. MS-102-3003) on MiSeq; (iii) OAS-95 samples: High Output Kit v2.5 300 Cycles (Illumina, Cat. No. 20024908) on NextSeq; (iv) CCI-55 samples: High Output Kit v2.5 150 Cycles (Illumina, Cat. No. 20024907) on NextSeq; (v) CCI-95 samples: Mid Output Kit v2.5 300 Cycles (Illumina, Cat. No. 20024905) on NextSeq.

The following steps were performed under a UV-decontaminated Laminar Flow PCR workbench (STARLAB International GmbH, Hamburg, Germany), sterilized with DNA AWAY (Thermo Fisher Scientific, Waltham, MA, USA). Prior to library preparation, the amplified DNA was cleaned with DNA Clean & Concentrator—5 (Zymo Research, Irvine, CA, USA). DNA input for library preparation was normalized to 5.98 ng µL⁻¹. Libraries were prepared using the NEBNext^® Ultra™ II FS DNA Library Prep Kit for Illumina (New England Biolabs (NEB), Ipswich, MA, USA), following the <100 ng input protocol. Fragmentation was set to 14 min and 7 PCR cycles were used. NEBNext^® Multiplex Oligos for Illumina^® were used for barcoding. Library concentration and size was quantified with Qubit™ DNA HS assay (Life Technologies, Carlsbad, CA, USA) and a Bioanalyzer High Sensitivity DNA kit (Agilent, Santa Clara, CA, USA). The libraries were sequenced using an Illumina NextSeq 550 with the High Output Kit v2.5 300 Cycles (2 × 150 bp paired-end) (Illumina, San Diego, CA, USA).