Smrt cell v3

SMRTbell libraries were constructed according to manufacturer’s instructions using the Greater Than 10 kb Template Preparation Using AMPure PB Beads and Sequencing (MagBead Station) protocols (Pacific Biosciences, Menlo Park, CA, USA). The extracted DNA was purified using a PowerClean DNA Clean-Up Kit (MoBio laboratories, Carlsbad, CA, USA). Small fragments of DNA were removed using AMPure PB Beads (Pacific Biosciences) of 0.45x. SMRTbell 20-kb libraries were prepared using a SMRTbell Template Prep Kit 1.0 (Pacific Biosciences). Libraries were subsequently sequenced on PacBio RS II (Pacific Biosciences) using DNA/Polymerase Binding Kit P6 v2 (Pacific Biosciences) and a DNA Sequencing Reagent Kit 4.0 (Pacific Biosciences). The titration density was 0.025 nM. The template was loaded into SMRT Cell v3 (Pacific Biosciences) using a Mag Bead Kit (Pacific Biosciences). Sequencing was performed using 30 cells in total. A 240-min movie was recorded for each cell. Metagenomic assembly was performed using a Hierarchical Genome Assembly Process (HGAP)^{27 (link)}. Minimus2 was used to attempt to overlap and join the resulting contigs^{28 (link)}. Final assemblies were achieved by polishing the draft assemblies using Quiver^{27 (link)}.

Details on preparation of reagents for the single-molecule assay are described in the Supplemental Information. Using a previously described protocol, the 30S pre-initiation complex (30S PIC) was prepared from Cy3B-labeled 30S subunit, ribosomal protein S1, initiation factor IF2, fMet-tRNA^fMet, biotinylated mRNA and 4 mM GTP. Prior to immobilizing the 30S PIC, we prepared the SMRT Cell v3 from Pacific Biosciences (Menlo Park), a ZMW chip, with Neutravidin. The 30S PIC was then diluted to 15 nM and loaded into the SMRT cell (22 (link)).
Ternary complexes (TCs) of Phe(Cy5)-tRNA^Phe and Ser-tRNAs were formed with EF-Tu (GTP) using a previously described protocol (23 (link)). After formation of TCs, a 2X delivery mix was created with 100–400 nM Cy5-TC^Phe and 400 nM-2 μM TC^Ser along with 200 nM BHQ-2-50S, 100 nM EF-G, 4 mM GTP, 2.5 mM Trolox and the oxygen scavenging system (PCA and PCD). Before starting an experiment, the delivery mix was diluted into polymix mixture in the SMRT Cell and loaded into a modified PacBio RSII sequencer (24 (link)). At the start of the experiment, the instrument illuminated the SMRT cell with a green laser and then transferred 20 μl of a delivery mixture onto the cell surface at t = 10 s. All experiments were performed at 20°C, and data was collected for 6 min.

The amount of primer and polymerase required for the binding reaction was determined using the SMRTbell concentration and library insert size. Primers were annealed and polymerase was bound to SMRTbell templates using the DNA/Polymerase Binding Kit P5 and P6 (Pacific Biosciences). Sequencing was performed using DNA sequencing reagent C3 and C4 (Pacific Biosciences) with Pacific Biosciences RSII sequencers and SMRT Cell V3 (Pacific Biosciences) at the University of Delaware Sequencing & Genotyping Center (DBI) and the Johns Hopkins University Deep Sequencing and Microarray Core (JHU). RSII loading efficiency was optimized for each individual library utilizing a standardized titration protocol. Over the course of the project, data capture time for the sequencing runs was initially set at 4 hr. This was extended to 6 hr after software upgrades.