DNA Polymerase II

The 16S rRNA gene was PCR-amplified with the primer pairs described above (details provided in Table S1) using Bullseye standard Taq DNA polymerase 2.0× master mix (MIDSCI, St. Louis, MO, USA). The PCR for each method was carried out in 50 µl reaction volumes in S1000 Thermal Cycler (BioRad, Hercules, CA, USA) with the following parameters: initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 40 s, 56°C for 1 min, and 72°C for 1 min with a final extension at 72°C for 10 min. PCR products were run on 1.5% agarose gel electrophoresis and the DNA band with the correct size was excised and purified using Wizard® SV Gel and PCR Clean-Up System (Promega, St. Louis, MO, USA). Equal amount of purified PCR products were pooled for subsequent 454 pyrosequencing.
454 pyrosequencing was carried out on the Titanium platform (Roche/454 Life Sciences) at the W.M. Keck Center, part of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. The barcoded and pooled amplicons were checked on an Agilent Bioanalzyer DNA7500 chip for the absence of primer-dimers, quantitated with Qubit assays (Invitrogen), and diluted to 1×10⁸ molecules/ul. Emulsion PCR was set up according to Roche's protocols for the three methods, each in duplicate. Sequencing was performed using 16-region gaskets and each sample was run in two lanes. Sequencing results were analyzed with Roche software version 2.5.3, signal processing for amplicons.

The gene encoding for Ssp from IFO-3046^{28 (link)} was codon optimised for expression in E. coli, synthesised and subcloned into pBAD/Myc-His-B by Invitrogen. Mutations to Ssp-pBAD were carried out using either the Stratagene QuikChange II method with Q5 High-Fidelity DNA polymerase (NEB, M0491S) and DpnI (NEB, R0176S) or the Q5 Site-Directed Mutagenesis Kit (NEB, E0552S). Primers used are detailed in Supplementary Table 5 and list of mutants generated in Supplementary Table 1. Successful mutations were verified by dideoxynucleotide sequencing (Macrogen, Korea).

Each DNA sequence coding for pri-miRNAs contained a DNA sequence coding for pre-miRNA and its 30 bp upstream and 25 bp downstream sequence in the C.elegans genomic DNA. Each pri-miRNA-coding DNA was added with a T7 promoter sequence at its 5p-end to generate a DNA template for in vitro transcription (IVT), so-called IVT-DNAs. The IVT-DNAs were synthesized by 2 methods. Twenty IVT-DNAs were produced by Phusion™ Hot Start II DNA Polymerase (Thermo Scientific) in the PCR reaction using a pair of primers and C.elegans genomic DNA or a synthetic ssDNA backbone as PCR template. One hundred and seventeen IVT-DNAs were made by Klenow Fragment (Thermo Scientific), which extended the partial dsDNAs generated from annealing pairs of synthetic ssDNAs. The primer sequences and synthesis methods for each IVT-DNA are shown in Supplementary Table S2. The pri-miRNAs were synthesized in a 20 μl IVT reaction mixture containing 200 ng IVT-DNA using the MEGAscript T7 Kit (Invitrogen). The IVT mixture was incubated at 37°C for 12 h. The IVT-DNA templates were then digested using TURBO DNase (Thermo Scientific). The reaction mixture was treated with 20 μl 2x TBE-Urea buffer and denatured at 75°C for 5 min. The denatured RNA was loaded onto a pre-run 10% Urea-PAGE. The RNA at the expected size was gel-purified and air-dried. Finally, the RNA was dissolved in distilled water and stored at -80°C for later use.

Meningococcal isolates were stored frozen (-80°C) at the NPHSL. For this study, isolates were cultured on Chocolate agar (Oxoid, UK) for 12-14 h at 36°C and 5% CO₂. DNA from cultured isolates was extracted using a GeneJET genomic DNA purification kit (Thermo Fisher Scientific, Vilnius, Lithuania) according to the manufacturer’s instructions. About 20 ng of purified chromosomal DNA was used as a template for amplification of all genes. Meningococcal isolates were serogrouped by slide agglutination by the isolate providers. Serogroup verification was performed by singleplex polymerase chain reaction (PCR)-based assay (Fraisier et al., 2009 ; Zhu et al., 2012 (link)). PCR was performed with Phire Green Hot Start II PCR Master Mix (Thermo Fisher Scientific, Vilnius, Lithuania) in 20-µL reaction volume comprising 0.4 µM of each primer (Metabion International AG, Germany). Amplification reactions included initial denaturation at 98°C for 30 s, 30 amplification cycles consisting of denaturation for 5 s at 98°C, annealing for 5 s at 60°C, and extension for 10 s at 72°C. These were followed by a final extension step for 1 min at 72°C. No template controls were included in the PCR assays. Amplification products were separated on 2.5% agarose gel.
MLST, PorA, and FetA typing for isolates received in 2009-2017 were performed according to the procedure described on the PubMLST Neisseria website (http://pubmlst.org/neisseria/; Jolley et al., 2018 (link)). The amplification enzymes used were Phire Hot Start II DNA polymerase included in the master mix (Thermo Fisher Scientific) in the case of porA and fetA, and DreamTaq Hot Start DNA polymerase included in the master mix (Thermo Fisher Scientific) for each of the MLST housekeeping genes. The reactions were performed in a 25-µL final volume. PCR conditions for amplification of porA and fetA were, as follows: initial denaturation at 98°C for 30 s, 30 amplification cycles consisting of denaturation for 5 s at 98°C, annealing for 5 s at 60°C, and extension for 15 s at 72°C. A final extension step lasted for 1 min at 72°C. Amplification reaction mixture with DreamTaq Hot Start DNA polymerase was subjected to initial denaturation at 95°C for 3 min, 35 cycles of denaturation at 95°C for 30 s, primer annealing at 57°C (for abcZ, adk, aroE, fumC, and pdhC) or 67°C (for pgm) for 30 s, extension at 72°C for 40 s, followed by a final extension step at 72°C for 7 min. Amplification products were analyzed on 1.5% agarose gel.
PCR products were purified using a DNA Clean & Concentrator kit (Zymo Research, Irvin, CA, US) and subjected to Sanger sequencing (Base Clear B.V., Leiden, The Netherlands). Allele assignment and MLST ST and cc were obtained from the PubMLST Neisseria database. For isolates received in 2017, amplification of vaccine antigens nhba, fhbp, and nadA was performed as previously described (Bambini et al., 2009 (link); Lucidarme et al., 2010 (link)). PCR reaction mixture with Phire Hot Start II DNA polymerase included in the master mix was subjected to initial denaturation at 98°C for 30 s, 30 amplification cycles consisting of denaturation for 5 s at 98°C, annealing for 5 s at 60°C (for nhba and nadA) or 63°C (for fhbp), extension for 15 s at 72°C, followed by a final extension step at 72°C for 1 min.
Internally directed primers nadaintF and nadaintR for detecting nadA were used as described by Lucidarme et al. (2009) (link). Briefly, PCR was performed with Phire Hot Start II DNA polymerase included in the master mix using reaction conditions described for amplification of porA and fetA.
For isolates not subjected to whole genome sequencing (WGS), the presence/absence of nadA in the meningococcal genome was confirmed using internally directed primers in case the application of primers targeting the flanking regions did not yield any PCR product (Lucidarme et al., 2009 (link)). The PubMLST Neisseria database was used to assign allelic and peptide variants of vaccine antigens.
Isolates received in 2018-2019 (n=25) underwent WGS with support from the European Center for Disease Prevention and Control. WGS was conducted by Eurofins Genomics Europe Sequencing Gmbh (Konstanz, Germany) using the Illumina NovaSeq platform. Assembly was performed using SPAdes 3.11 (Bankevich et al., 2012 (link)) with careful mode enabled. Genome contigs were submitted to the PubMLST Neisseria database (see Data Availability Statement). The allelic profile of seven MLST genes and variable regions of porA and fetA were determined by automatic scanning of genome contigs (Jolley et al., 2018 (link)). Allelic and peptide variants of vaccine antigens (fhbp, nhba, and nadA) were also defined.