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      MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island.

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          Abstract

          Short-read, high-throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes. The MinION nanopore sequencer can produce long sequencing reads on a device similar in size to a USB memory stick. Here we apply a MinION sequencer to resolve the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58. Nanopore sequencing data from a single 18-h run was used to create a scaffold for an assembly generated from short-read Illumina data. Our results demonstrate the potential of the MinION device in clinical laboratories to fully characterize the epidemic spread of bacterial pathogens.

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          Most cited references20

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          Continuous base identification for single-molecule nanopore DNA sequencing.

          A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications.
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            Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18.

            Salmonella enterica serovar Typhi (S. typhi) is the aetiological agent of typhoid fever, a serious invasive bacterial disease of humans with an annual global burden of approximately 16 million cases, leading to 600,000 fatalities. Many S. enterica serovars actively invade the mucosal surface of the intestine but are normally contained in healthy individuals by the local immune defence mechanisms. However, S. typhi has evolved the ability to spread to the deeper tissues of humans, including liver, spleen and bone marrow. Here we have sequenced the 4,809,037-base pair (bp) genome of a S. typhi (CT18) that is resistant to multiple drugs, revealing the presence of hundreds of insertions and deletions compared with the Escherichia coli genome, ranging in size from single genes to large islands. Notably, the genome sequence identifies over two hundred pseudogenes, several corresponding to genes that are known to contribute to virulence in Salmonella typhimurium. This genetic degradation may contribute to the human-restricted host range for S. typhi. CT18 harbours a 218,150-bp multiple-drug-resistance incH1 plasmid (pHCM1), and a 106,516-bp cryptic plasmid (pHCM2), which shows recent common ancestry with a virulence plasmid of Yersinia pestis.
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              Is Open Access

              Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition

              We characterize and extend a highly efficient method for constructing shotgun fragment libraries in which transposase catalyzes in vitro DNA fragmentation and adaptor incorporation simultaneously. We apply this method to sequencing a human genome and find that coverage biases are comparable to those of conventional protocols. We also extend its capabilities by developing protocols for sub-nanogram library construction, exome capture from 50 ng of input DNA, PCR-free and colony PCR library construction, and 96-plex sample indexing.
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                Author and article information

                Journal
                Nat. Biotechnol.
                Nature biotechnology
                1546-1696
                1087-0156
                Mar 2015
                : 33
                : 3
                Affiliations
                [1 ] Gastrointestinal Bacteria Reference Unit, Public Health England, Colindale, London, UK.
                [2 ] 1] Department of Biomedical Sciences, University of Sassari, Sassari, Italy. [2] Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
                [3 ] National Reference Centre for Salmonellae and other Enterics, Robert Koch Institute, Wernigerode, Germany.
                [4 ] Norwich Medical School, University of East Anglia, Norwich, UK.
                Article
                nbt.3103
                10.1038/nbt.3103
                25485618
                4c753888-7a26-4e7c-a1ec-1ef9eac106c8
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