Recent advances in long read technologies not only enable large consortia to aim to sequence all eukaryotes on Earth, but they also allow individual laboratories to sequence their species of interest with relatively low investment. Long read technologies embody the promise of overcoming scaffolding problems associated with repeats and low complexity sequences, but the number of contigs often far exceeds the number of chromosomes and they may contain many insertion and deletion errors around homopolymer tracts. To overcome these issues, we have implemented the ILRA pipeline to correct long read-based assemblies. Contigs are first reordered, renamed, merged, circularized, or filtered if erroneous or contaminated. Illumina short reads are used subsequently to correct homopolymer errors. We successfully tested our approach by improving the genome sequences of Homo sapiens, Trypanosoma brucei, and Leptosphaeria spp., and by generating four novel Plasmodium falciparum assemblies from field samples. We found that correcting homopolymer tracts reduced the number of genes incorrectly annotated as pseudogenes, but an iterative approach seems to be required to correct more sequencing errors. In summary, we describe and benchmark the performance of our new tool, which improved the quality of novel long read assemblies up to 1 Gbp. The pipeline is available at GitHub: https://github.com/ThomasDOtto/ILRA.

The global spread of high-risk clo1nes (HRCs) of multidrug-resistant (MDR) Pseudomonas aeruginosa has hindered infection control and treatment strategies worldwide. In Colombia, globally relevant HRCs such as ST235 and ST111 have been widely reported. In this study, we evaluated phenotypic and metabolic variations associated with intracellular survival and dissemination in P. aeruginosa. A total of 100 clinical isolates were collected from 22 hospitals in Colombia. The isolates had been previously characterized and classified as MDR or susceptible strains (SSs), and their sequence types (STs) had been earlier determined. Based on this prior characterization, isolates were grouped in this study as multidrug-resistant high-risk clones (HRC, n = 50; corresponding to sequence types ST235 and ST111), multidrug-resistant non-high-risk clones (NHRCs, n = 27; non-ST235/ST111), and susceptible strains (SS, n = 23; also, non-ST235/ST111). Phenotypic traits, including motility, spontaneous mutation frequency, biofilm formation, and pigment production, were evaluated. In addition, a subset of 30 isolates was assessed for intracellular survival in vitro and metabolomic profiling using liquid chromatography-mass spectrometry. HRC isolates exhibited significantly reduced motility compared with NHRC and SS isolates (swarming: HRC vs. NHRC, p = 0.0032; HRC vs. SS, p = 0.010; swimming: HRC vs. NHRC and SS, p < 0.0001; twitching: HRC vs. SS, p = 0.0004), as well as lower pigment production (pyocyanin: HRC vs. NHRC and SS, p < 0.0001; pyoverdine: HRC vs. NHRC, p < 0.0001). Metabolomic analysis revealed increased concentrations of metabolites associated with iron acquisition and siderophore-related pathways in HRC isolates. Overall, these findings suggest that P. aeruginosa HRCs display distinct phenotypic and metabolic patterns that may contribute to persistence and dissemination in clinical settings, contributing to their epidemiological success.