Smrtportal analysis platform

Chromosomal DNA from B. longum subsp. longum NCIMB 8809 and B. longum subsp. longum CCUG 30698 was isolated as previously described [58 ] and purified using the PowerClean DNA Clean-Up Kit by MoBio Laboratories (Carlsbad, CA). SMRT bell library preparation was performed as previously described [59 (link), 60 (link)]. SMRT sequencing was performed on a PacBio RS instrument (executed by GATC Biotech, Germany) and assembled using the Pacific Biosciences SMRTPortal analysis platform (version 2.1.1). Illumina sequencing was performed by the commercial sequencing service providers Macrogen (Seoul, Republic of Korea) (using a paired-end library). The Illumina sequences obtained were then assembled with the SMRTPortal output using MIRA v3.9 (http://www.chevreux.org/projects_mira.html). Remaining gaps and quality issues were resolved using Sanger sequencing of PCR products.
To identify methylated positions the Pacific Biosciences SMRTPortal analysis platform (version 1.4) was adopted, this employs an in silico kinetic reference and a t-test based kinetic score detection of modified base positions.

Reads were mapped against the P12 genome and plasmid sequences (accession numbers CP001217 and CP001218, respectively). The ModH5 methylation recognition site was identified using the Pacific Biosciences’ SMRTPortal analysis platform (v. 1.3.1) as described previously^{54 (link)}, and its locations relative to genome features analysed using Artemis^{55 (link)} and in-house scripts written in Perl and Python. Circular genome figures were created using DNAPlotter^{56 (link)} using data derived from the Prokka annotation and SMRT methylome. Comparative analysis of tetranucleotide extremes in H. pylori genomes was performed using the Signature server (Institute of Bioinformatics, University of Georgia; http://www.cmbl.uga.edu/software/signature.html) to determine Karlin’s tau (τ_wxyz) values whereby values less than 0.72 or greater than 1.28 indicate significantly underrepresented or overrepresented tetranucleotide motifs, respectively. Statisitcal analysis of GACC prevalence and motility was performed using GraphPad Prism software (v6.0 h).

Sequencing was performed utilizing a combined SMRT sequencing and Illumina approach on a Pacific Biosciences RS II sequencing platform (executed by GATC Biotech Ltd., Germany) and an Illumina MiSeq platform (executed by GenProbio s.r.l., Parma, Italy). De novo assemblies were performed on the Pacific Biosciences SMRTPortal analysis platform (version 2.3.1), utilizing the RS_HGAP_Assembly.2 protocol. Hybrid assemblies were performed utilizing the Unicycler hybrid assembly pipeline (Wick et al., 2017 (link)). Remaining low quality regions and sequencing conflicts were resolved by primer walking and Sanger sequencing of PCR products (performed by Eurofins MWG Operon, Germany). Open Reading Frame (ORF) prediction was performed with Prodigal v2.5 prediction software (Hyatt et al., 2010 (link)) and confirmed using BLASTX v2.2.26 alignments (Altschul et al., 1990 (link)). ORFs were automatically annotated using BLASTP v2.2.26 (Altschul et al., 1990 (link)) analysis against the non-redundant protein databases curated by the National Centre for Biotechnology Information (NCBI). Artemis v18 genome browser and annotation tool was used to manually curate ORFs and for the combination and inspection of ORF results. Final ORF annotations were refined where necessary using alternative databases; Pfam (Bateman et al., 2004 (link)), HHpred (Söding et al., 2005 (link)), and Uniprot/EMBL.