A comparison of Oxford nanopore library strategies for bacterial genomics

Sauvage, Thomas; Cormier, Alexandre; Delphine, Passerini

doi:10.1186/s12864-023-09729-z

Cited by 5 publications

(1 citation statement)

References 41 publications

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“…However, advancements in ONT's V14 chemistry, combined with the R10.4.1 pore, and enhanced by Dorado's high-precision basecalling models (SUP, v4.2.0), have significantly improved accuracy. This progress 15,16 established ONT's MinIon as a dependable option for sequencing in parSEQ.…”

Section: Choice Of Sequencing Platformmentioning

confidence: 99%

parSEQ:Probe and Rescue Sequencing for Advanced Variant Retrieval from DNA Pool

Houmani,

Peterkin,

Antoun

et al. 2023

Preprint

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Modern protein engineering is powered by sequence-function data-sets. We have developed parSEQ, a platform that maximizes the capture of these protein sequence-function data-sets through a sequence-first-screen-later approach. parSEQ relies on Next-Generation Sequencing (NGS) to reverse the conventional screen-first-sequence-later workflow. This allows for the high-throughput retrieval of variant sequences from DNA pools, ensuring that every screened variant’s functional data is paired with its sequence data. This report details parSEQ’s methodology and its integration into various protein engineering workflows. Through several case studies, we illustrate parSEQ’s broad applicability. These case studies describe the use of parSEQ for high-throughput variant DNA-template retrieval, expression-ready bacterial clone isolation, sourcing DNA for denovo designed proteins, and preparing targeted mutational libraries. Our findings suggest that parSEQ’s approach to capturing sequence-function data-sets can advance protein engineering efforts, particularly in the age of artificial intelligence and machine learning.

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Section: Choice Of Sequencing Platformmentioning

confidence: 99%

parSEQ:Probe and Rescue Sequencing for Advanced Variant Retrieval from DNA Pool

Houmani,

Peterkin,

Antoun

et al. 2023

Preprint

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Extraction of high‐molecular‐weight DNA from Streptococcus spp. for nanopore sequencing in resource‐limited settings

Das,

Delamare‐Deboutteville,

Barnes

et al. 2024

MicrobiologyOpen

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The long‐read sequencing platform MinION, developed by Oxford Nanopore Technologies, enables the sequencing of bacterial genomes in resource‐limited settings, such as field conditions or low‐ and middle‐income countries. For this purpose, protocols for extracting high‐molecular‐weight DNA using nonhazardous, inexpensive reagents and equipment are needed, and some methods have been developed for gram‐negative bacteria. However, we found that without modification, these protocols are unsuitable for gram‐positive Streptococcus spp., a major threat to fish farming and food security in low‐ and middle‐income countries. Multiple approaches were evaluated, and the most effective was an extraction method using lysozyme, sodium dodecyl sulfate, and proteinase K for lysis of bacterial cells and magnetic beads for DNA recovery. We optimized the method to consistently achieve sufficient yields of pure high‐molecular‐weight DNA with minimal reagents and time and developed a version of the protocol which can be performed without a centrifuge or electrical power. The suitability of the method was verified by MinION sequencing and assembly of 12 genomes of epidemiologically diverse fish‐pathogenic Streptococcus iniae and Streptococcus agalactiae isolates. The combination of effective high‐molecular‐weight DNA extraction and MinION sequencing enabled the discovery of a naturally occurring 15 kb low‐copy number mobilizable plasmid in S. iniae, which we name pSI1. We expect that our resource‐limited settings‐adapted protocol for high‐molecular‐weight DNA extraction could be implemented successfully for similarly recalcitrant‐to‐lysis gram‐positive bacteria, and it represents a method of choice for MinION‐based disease diagnostics in low‐ and middle‐income countries.

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Rich microbial and depolymerising diversity in Antarctic krill gut

Möller,

Vainshtein,

Meyer

et al. 2024

Microbiol Spectr

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With almost a quadrillion individuals, the Antarctic krill processes five million tons of organic carbon every day during austral summer. This high carbon flux requires a broad range of hydrolytic enzymes to decompose the diverse food-derived biopolymers. While krill itself possesses numerous such enzymes, it is unclear, to what extent the endogenous microbiota contribute to the hydrolytic potential of the gut environment. Here we applied amplicon sequencing, shotgun metagenomics, cultivation, and physiological assays to characterize the krill gut microbiota. The broad bacterial diversity (273 families, 919 genera, and 2,309 species) also included a complex potentially anaerobic sub-community. Plate-based assays with 198 isolated pure cultures revealed widespread capacities to utilize lipids (e.g., tributyrin), followed by proteins (casein) and to a lesser extent by polysaccharides (e.g., alginate and chitin). While most isolates affiliated with the genera Pseudoalteromonas and Psychrobacter , also Rubritalea spp. (Verrucomicrobia) were observed. The krill gut microbiota growing on marine broth agar plates possess 13,012 predicted hydrolyses; 15-fold more than previously predicted from a transcriptome-proteome compendium of krill. Cultivation-independent and -dependent approaches indicated members of the families Flavobacteriaceae and Pseudoalteromonadaceae to dominate the capacities for lipid/protein hydrolysis and to provide a plethora of carbohydrate-active enzymes, sulfatases, and laminarin- or porphyrin-depolymerizing hydrolases. Notably, also the potential to hydrolyze plastics such as polyethylene terephthalate and polylactatide was observed, affiliating mostly with Moraxellaceae. Overall, this study shows extensive microbial diversity in the krill gut, and suggests that the microbiota likely play a significant role in the nutrient acquisition of the krill by enriching its hydrolytic enzyme repertoire. IMPORTANCE The Antarctic krill ( Euphausia superba ) is a keystone species of the Antarctic marine food web, connecting the productivity of phyto- and zooplankton with the nutrition of the higher trophic levels. Accordingly, krill significantly contributes to biomass turnover, requiring the decomposition of seasonally varying plankton-derived biopolymers. This study highlights the likely role of the krill gut microbiota in this ecosystem function by revealing the great number of diverse hydrolases that microbes contribute to the krill gut environment. The here resolved repertoire of hydrolytic enzymes could contribute to the overall nutritional resilience of krill and to the general organic matter cycling under changing environmental conditions in the Antarctic sea water. Furthermore, the krill gut microbiome could serve as a valuable resource of cold-adapted hydrolytic enzymes for diverse biotechnological applications.

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A comparison of Oxford nanopore library strategies for bacterial genomics

Cited by 5 publications

References 41 publications

parSEQ:Probe and Rescue Sequencing for Advanced Variant Retrieval from DNA Pool

parSEQ:Probe and Rescue Sequencing for Advanced Variant Retrieval from DNA Pool

Extraction of high‐molecular‐weight DNA from Streptococcus spp. for nanopore sequencing in resource‐limited settings

Rich microbial and depolymerising diversity in Antarctic krill gut

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