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Fast Identification Method for Screening Bacteria from Faecal Samples Using Oxford Nanopore Technologies MinION Sequencing.

ABSTRACT: Most bacterial identification methods require extensive culturing, strain purification and DNA extraction protocols. This leads to additional expenses and time lags when isolating specific bacteria from complex microbiological ecosystems. This study aimed to develop a fast and robust method for identification of lactobacilli, bifidobacteria and Bacteroides in human faecal samples. Bacteria from faecal samples were cultured anaerobically on selective media. Sonication-based DNA extraction was performed, followed by almost complete 16S rRNA gene polymerase chain reaction amplification and MinION sequencing with the Flongle adapter. Sequence analysis was performed using NanoCLUST, while RStudio was used for graphics. For 110 of the 125 colonies investigated, 100% of reads were attributed to a single species, while the remaining 15 colonies consisted of mixtures of up to three different species. The proposed bacterial identification method is advantageous for isolating particular bacteria for which there are no exclusively selective media, as it avoids lengthy colony purification and DNA purification methods, and yields a quick colony identification with high accuracy. Therefore, this method can be used for directly screening for pure cultures of target microorganisms and is suitable for the identification of bacteria in culturomics studies.

SUBMITTER: Borges ASG

PROVIDER: S-EPMC9911510 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Fast Identification Method for Screening Bacteria from Faecal Samples Using Oxford Nanopore Technologies MinION Sequencing.

Borges Ana Sofia G ASG Basu Meghna M Brinks Erik E Bang Corinna C Cho Gyu-Sung GS Baines John F JF Franke Andre A Franz Charles M A P CMAP

Current microbiology 20230209 3

Most bacterial identification methods require extensive culturing, strain purification and DNA extraction protocols. This leads to additional expenses and time lags when isolating specific bacteria from complex microbiological ecosystems. This study aimed to develop a fast and robust method for identification of lactobacilli, bifidobacteria and Bacteroides in human faecal samples. Bacteria from faecal samples were cultured anaerobically on selective media. Sonication-based DNA extraction was per ...[more]

PMID: 36759384

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Project description:BackgroundSequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications.ResultsHere, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies' MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing.ConclusionsOur pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.

Project description:Sequencing DNA directly from patient samples enables faster pathogen characterization compared to traditional culture-based approaches, but often yields insufficient sequence data for effective downstream analysis. CRISPR-Cas9 enrichment is designed to improve the yield of low abundance sequences but has not been thoroughly explored with Oxford Nanopore Technologies (ONT) for use in clinical bacterial epidemiology. We designed CRISPR-Cas9 guide RNAs to enrich the human pathogen Klebsiella pneumoniae, by targeting multi-locus sequence type (MLST) and transfer RNA (tRNA) genes, as well as common antimicrobial resistance (AMR) genes and the resistance-associated integron gene intI1. We validated enrichment performance in 20 K. pneumoniae isolates, finding that guides generated successful enrichment across all conserved sites except for one AMR gene in two isolates. Enrichment of MLST genes led to a correct allele call in all seven loci for 8 out of 10 isolates that had depth of 30× or more in these regions. We then compared enriched and unenriched sequencing of three human fecal samples spiked with K. pneumoniae at varying abundance. Enriched sequencing generated 56× and 11.3× the number of AMR and MLST reads, respectively, compared to unenriched sequencing, and required approximately one-third of the computational storage space. Targeting the intI1 gene often led to detection of 10-20 proximal resistance genes due to the long reads produced by ONT sequencing. We demonstrated that CRISPR-Cas9 enrichment combined with ONT sequencing enabled improved genomic characterization outcomes over unenriched sequencing of patient samples. This method could be used to inform infection control strategies by identifying patients colonized with high-risk strains.ImportanceUnderstanding bacteria in complex samples can be challenging due to their low abundance, which often results in insufficient data for analysis. To improve the detection of harmful bacteria, we implemented a technique aimed at increasing the amount of data from target pathogens when combined with modern DNA sequencing technologies. Our technique uses CRISPR-Cas9 to target specific gene sequences in the bacterial pathogen Klebsiella pneumoniae and improve recovery from human stool samples. We found our enrichment method to significantly outperform traditional methods, generating far more data originating from our target genes. Additionally, we developed new computational techniques to further enhance the analysis, providing a thorough method for characterizing pathogens from complex biological samples.

Dataset Information

Fast Identification Method for Screening Bacteria from Faecal Samples Using Oxford Nanopore Technologies MinION Sequencing.

Publications

Fast Identification Method for Screening Bacteria from Faecal Samples Using Oxford Nanopore Technologies MinION Sequencing.

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