ZymoCSII_D6310_5kb
Library originated from aliquots of a ZymoBIOMICS microbial community standard (CSII) containing ~1.5 x 109 cells / mL of 10 microbial species (8 bacterial and 2 yeast species, genomes available at: https://s3.amazonaws.com/zymo-files/BioPool/ZymoBIOMICS.STD.refseq.v2.zip) in log-distributed abundance ranging from 89.1% (Listeria monocytogenes) to 89 x 10-6 % (Staphylococcus aureus). After dividing the standard into numerous 75 l aliquots, the mock community aliquots of gDNA were extracted using mechanical and chemical lysis and column-based purification and QC. Multiple aliquots of gDNA were combined to meet DNA input requirements for each specific DNA fragment size shearing protocol. Each composited gDNA sample was then subjected to mechanical shearing protocols to achieve either ~2, ~5, or ~8 kb fragment sizes via sonication (for 2 and 5 kb) or hydrodynamic centrifugation (for ~8 kb). Following gel-based size selection and bead-based clean-up, each mock microbial standard sample was subjected to probe-based enrichment and library construction utilizing modified and customized procedures adapted from methods of the SureSelectXT V1 platform and custom biotinylated probes. Following these steps, libraries were further subjected to PacBio library creation using PacBio SMRTbell Express Template Preparation Kit without any additional shearing or amplification. Following hairpin adapter ligation, all enriched libraries for circular consensus sequencing were cleaned using nuclease treatment and AMPure PB bead clean-up.