DNA fragmentation and quality control analysis using Diagenode shearing systems and Fragment Analyzer

Lakha, Wassim; Panteleeva, Irina; Squazzo, Sharon L.; Saxena, Rini; Kroonen, Jerome; Siembieda, Steve; Tilmes, Markus; Hagopian, Jonathan C.

doi:10.1038/nmeth.f.397

Cited by 4 publications

(4 citation statements)

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“…The SPRI bead cleanup and Circulomics Short Read Eliminator protocols favoured size selection and successfully removed short fragments of DNA (∼4kbp) appropriate for long read sequencing to prevent fuel usage in flow cells, although short DNA removal may lead to the loss of resistance plasmids 35 . DNA shearing by g-tubes technique produced DNA fragments suitable for sequencing long read ligation library preparations 36,37…”

Section: Discussionmentioning

confidence: 99%

“…The SPRI bead cleanup and Circulomics Short Read Eliminator protocols favoured size selection and successfully removed short fragments of DNA (~4kbp) appropriate for long read sequencing to prevent fuel usage in flow cells, although short DNA removal may lead to the loss of resistance plasmids 35 . DNA shearing by g-tubes technique produced DNA fragments suitable for sequencing long read ligation library preparations 36,37 Plasmid recovery from the extraction methods was assessed by downstream sequencing of a fully annotated reference genome (E. coli EC958) which contains two plasmids 27 . The QIAamp Mini-pure enzyme-based extraction will not cleave plasmids and in turn, providing no ends for ligation library preparation 35 .…”

Section: Discussionmentioning

confidence: 99%

“…The QIAamp Mini-pure enzyme-based extraction will not cleave plasmids and in turn, providing no ends for ligation library preparation 35 . Although not undertaken, DNA shearing by g-tubes or megaruptor techniques produce DNA fragments suitable for sequencing library preparations 36,37 . The UltraPure method using the mechanical and chemical lysis was effective at producing optimal DNA lengths with available ends.…”

Section: Discussionmentioning

confidence: 99%

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An optimised method for bacterial nucleic acid extraction from positive blood culture broths for whole genome sequencing, resistance phenotype prediction and downstream molecular applications

Bauer

Peri

Lüftinger

et al. 2022

Preprint

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Background: A prerequisite to rapid molecular detection of pathogens causing bloodstream infections is an efficient, cost effective and robust DNA extraction solution. We describe methods for microbial DNA extraction direct from positive blood culture broths, suitable for metagenomic sequencing and the application of machine-learning based tools to predict antimicrobial susceptibility. Methods: Prospectively collected culture-positive blood culture broths with Gram-negative bacteria, were directly extracted using various commercially available kits. We compared methods for efficient inhibitor removal, avoidance of DNA shearing or degradation, to achieve DNA of high quality and purity. Bacterial species identified via whole-genome metagenomic sequencing (Illumina, MiniSeq) from blood culture extracts were compared to conventional methods from cultured isolates (Vitek MS). A machine-learning algorithm (AREScloud) was used to predict susceptibility against commercially available antibiotics, compared to susceptibility testing (Vitek 2) and other commercially available rapid diagnostic instruments (Accelerate Pheno and BCID). Results: A two-kit method using a modified MolYsis Basic kit (for host DNA depletion) and extraction using Qiagen DNeasy UltraClean microbial kits resulted in optimal extractions appropriate for multiple molecular applications, including PCR, short-read and long-read sequencing. DNA extracts from 40 blood culture broths were included. Taxonomic profiling by direct metagenomic sequencing matched species identification by conventional methods in 38/40 (95%) of samples, with two showing agreement to genus level. In two polymicrobial samples, a second organism was missed by sequencing. Whole genome sequencing antimicrobial susceptibility testing (WGS-AST) models were able to accurately infer profiles for 6 common pathogens against 17 antibiotics. Overall categorical agreement (CA) was 95%, with 11% very major errors (VME) and 3.9% major errors (ME). CA for WGS-AST was >95% for 5/6 of the most common pathogens (E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and C. jejuni) while it was lower for K. oxytoca (66.7%), likely due to the presence of inducible cephalosporinases. Performance of WGS-AST was sub-optimal for uncommon pathogens (e.g. Elizabethkingia) and some combination antibiotic compounds (e.g. ticarcillin-clavulanate). Time to pathogen identification and resistance gene detection was fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a rapid MIC result in approximately 8 h. While Illumina based direct metagenomic sequencing did not result in faster turn-around times compared conventional methods, use of real-time nanopore sequencing may allow faster data acquisition. Conclusions: The application of direct metagenomic sequencing from positive blood culture broths is a feasible approach and solves some of the challenges of sequencing from low-bacterial load samples. Machine-learning based algorithms are also accurate for common pathogen / drug combinations, although additional work is required to optimise algorithms for uncommon species and more complex resistance genotypes, as well as streamlining methods to provide more rapid sequencing results.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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An optimised method for bacterial nucleic acid extraction from positive blood culture broths for whole genome sequencing, resistance phenotype prediction and downstream molecular applications

Bauer

Peri

Lüftinger

et al. 2022

Preprint

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“…The restriction fragments were mapped on 0.8% agarose gel and stained with red staining dye ( Szymczak et al, 2020 ). For further characterization of DNA, electropherogram of EcoRI digested DNA was mapped with Agilent Fragment Analyzer Systems (Hangzhou Lianchuan Biotechnology Co., Ltd., China) to estimate the genome size ( Lakha et al, 2016 ; Yu et al, 2016 ).…”

Section: Methodsmentioning

confidence: 99%

Virulent Drexlervirial Bacteriophage MSK, Morphological and Genome Resemblance With Rtp Bacteriophage Inhibits the Multidrug-Resistant Bacteria

et al. 2021

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Phage-host interactions are likely to have the most critical aspect of phage biology. Phages are the most abundant and ubiquitous infectious acellular entities in the biosphere, where their presence remains elusive. Here, the novel Escherichia coli lytic bacteriophage, named MSK, was isolated from the lysed culture of E. coli C (phix174 host). The genome of phage MSK was sequenced, comprising 45,053 bp with 44.8% G + C composition. In total, 73 open reading frames (ORFs) were predicted, out of which 24 showed a close homology with known functional proteins, including one tRNA-arg; however, the other 49 proteins with no proven function in the genome database were called hypothetical. Electron Microscopy and genome characterization have revealed that MSK phage has a rosette-like tail tip. There were, in total, 46 ORFs which were homologous to the Rtp genome. Among these ORFs, the tail fiber protein with a locus tag of MSK_000019 was homologous to Rtp 43 protein, which determines the host specificity. The other protein, MSK_000046, encodes lipoprotein (cor gene); that protein resembles Rtp 45, responsible for preventing adsorption during cell lysis. Thirteen MSK structural proteins were identified by SDS-PAGE analysis. Out of these, 12 were vital structural proteins, and one was a hypothetical protein. Among these, the protein terminase large (MSK_000072) subunit, which may be involved in DNA packaging and proposed packaging strategy of MSK bacteriophage genome, takes place through headful packaging using the pac-sites. Biosafety assessment of highly stable phage MSK genome analysis has revealed that the phage did not possess virulence genes, which indicates proper phage therapy. MSK phage potentially could be used to inhibit the multidrug-resistant bacteria, including AMP, TCN, and Colistin. Further, a comparative genome and lifestyle study of MSK phage confirmed the highest similarity level (87.18% ANI). These findings suggest it to be a new lytic isolated phage species. Finally, Blast and phylogenetic analysis of the large terminase subunit and tail fiber protein put it in Rtp viruses’ genus of family Drexlerviridae.

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Optimized Method for Bacterial Nucleic Acid Extraction from Positive Blood Culture Broth for Whole-Genome Sequencing, Resistance Phenotype Prediction, and Downstream Molecular Applications

et al. 2022

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DNA fragmentation and quality control analysis using Diagenode shearing systems and Fragment Analyzer

Cited by 4 publications

References 1 publication

An optimised method for bacterial nucleic acid extraction from positive blood culture broths for whole genome sequencing, resistance phenotype prediction and downstream molecular applications

An optimised method for bacterial nucleic acid extraction from positive blood culture broths for whole genome sequencing, resistance phenotype prediction and downstream molecular applications

Virulent Drexlervirial Bacteriophage MSK, Morphological and Genome Resemblance With Rtp Bacteriophage Inhibits the Multidrug-Resistant Bacteria

Optimized Method for Bacterial Nucleic Acid Extraction from Positive Blood Culture Broth for Whole-Genome Sequencing, Resistance Phenotype Prediction, and Downstream Molecular Applications

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