Dataset Information

DNA enrichment and tagmentation method for species-level identification and strain-level differentiation using ON-rep-seq.

ABSTRACT: Despite the massive developments within culture-independent methods for detection of microorganisms during the last decade, culture-based methods remain a cornerstone in microbiology. Yet, the problem of rapid, accurate and inexpensive identification of bacterial isolates down to species/strain level remains unresolved. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (<24?h) and cost-effective species level identification and strain level differentiation using the MinION portable sequencing platform (ON-rep-seq). DNA library preparation for 96 isolates takes less than 5?h and ensures highly reproducible distribution of reads that can be used to generate strain level specific read length counts profiles (LCp). We have developed a pipeline that by correcting reads error within peaks of LCp generates a set of high quality (>99%) consensus reads. Whereas, the information from high quality reads is used to retrieve species level taxonomy, comparison of LCp allows for strain level differentiation.

SUBMITTER: Krych L

PROVIDER: S-EPMC6787052 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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DNA enrichment and tagmentation method for species-level identification and strain-level differentiation using ON-rep-seq.

Krych Łukasz Ł Castro-Mejía Josué L JL Forero-Junco Laura M LM Moesby Daniel N DN Mikkelsen Morten B MB Rasmussen Morten A MA Sykulski Maciej M Nielsen Dennis S DS

Communications biology 20191010

Despite the massive developments within culture-independent methods for detection of microorganisms during the last decade, culture-based methods remain a cornerstone in microbiology. Yet, the problem of rapid, accurate and inexpensive identification of bacterial isolates down to species/strain level remains unresolved. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (<24 h) and cost-effective species level identification and strain level differentiatio ...[more]

PMID: 31633060

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Project description:BackgroundPreviously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method's flexibility to a broader range of viruses, both DNA and RNA.ResultsCompared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run.ConclusionTaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses.

Project description:The Achromobacter is a genus in the family Alcaligenaceae, comprising fifteen species isolated from different sources, including clinical samples. The ability to detect and correctly identify Achromobacter species, particularly A. xylosoxidans, and differentiate them from other phenotypically similar and genotypically related Gram-negative, aerobic, non-fermenting species is important for patients with cystic fibrosis (CF), as well as for nosocomial and other opportunistic infections. Traditional phenotypic profile-based analyses have been demonstrated to be inadequate for reliable identifications of isolates of Achromobacter species and genotypic-based assays, relying upon comparative 16S rRNA gene sequence analyses are not able to insure definitive identifications of Achromobacter species, due to the inherently conserved nature of the gene. The uses of alternative methodologies to enable high-resolution differentiation between the species in the genus are needed. A comparative multi-locus sequence analysis (MLSA) of four selected 'house-keeping' genes (atpD, gyrB, recA, and rpoB) assessed the individual gene sequences for their potential in developing a reliable, rapid and cost-effective diagnostic protocol for Achromobacter species identifications. The analysis of the type strains of the species of the genus and 46 strains of Achromobacter species showed congruence between the cluster analyses derived from the individual genes. The MLSA gene sequences exhibited different levels of resolution in delineating the validly published Achromobacter species and elucidated strains that represent new genotypes and probable new species of the genus. Our results also suggested that the recently described A. spritinus is a later heterotypic synonym of A. marplatensis. Strains were analyzed, using whole-cell Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS), as an alternative phenotypic profile-based method with the potential to support the identifications determined by the genotypic DNA sequence-based MLSA. The MALDI-TOF MS data showed good accordance in strain groupings and identifications by the MLSA data.

Dataset Information

DNA enrichment and tagmentation method for species-level identification and strain-level differentiation using ON-rep-seq.

Publications

DNA enrichment and tagmentation method for species-level identification and strain-level differentiation using ON-rep-seq.

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