Cluster generation and sequencing: Pooled libraries are denatured and diluted to the appropriate clustering concentration. Denatured and diluted libraries are loaded onto the NextSeq 2000 cartridge and clustering occurs onboard the instrument. Once clustering is complete, sequencing immediately commences using Illumina’s 2-color SBS chemistry. Upon completion of read 1, an index read 1 of varying length is performed depending on the library kit used. If libraries are dual indexed, a second index read is performed. Finally, the library fragments are re-synthesized in reverse orientation and sequenced from the opposite end of the read 1 fragment to produce the paired end read 2.