LabMedica International
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Pneumonia Diagnosed by Nanopore Sequencing of Sputum athogen identification in patients with community-acquired pneumonia primarily relies on culture-based techniques. Sequencing-based approaches for pathogen identification are being applied to pneumonia patients. Haemophilus influenzae, a type of bacteria, can cause many different kinds of infections. These infections range from mild ear infections to severe diseases, like bloodstream infections. H. influenzae is an opportunistic pathogen of the respiratory tract that becomes pathogenic only when other risk factors are present. Scientists at Seoul National University Hospital (Seoul, South Korea; www.snuh.org) and their colleagues used deep sequencing of the 16S rRNA gene from sputum to identify H. influenza in a patient with community-acquired pneumonia. They extracted genomic DNA (Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA; www.thermofisher.com) from sputum obtained by oropharyngeal suction after a single empiric administration of an antimicrobial drug (cefuroxime, 500 mg). They generated the sequencing libraries using a rapid 16S amplicon sequencing kit. The team retrospectively performed 16S amplicon sequencing with MinION (Oxford Nanopore Technologies, Oxford, UK; www.nanoporetech.com), a nanopore sequencer, that is gaining attention in metagenomics studies because of its capability for long-read sequencing and real-time analysis, along with its small size. They identified the pneumonia pathogen in this patient by deep sequencing of 16S amplicons from sputum using MinION. The reads aligned to H. influenzae were >100-fold more abundant than reads aligned with other commensal bacteria, reflecting the significant proliferation of H. influenzae in the patient’s respiratory tract. The authors concluded that with the MinION sequencer, generated
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reads can be analyzed in real time, which makes this approach more promising. Tentative point-of-care diagnosis by nanopore 16S sequencing and confirmation of the result by standard culture methods would be a feasible approach. They performed sequencing for five hours and the subgroup analyses of reads generated for the first hour and for the first 10 minutes produced similar results, indicating that a relatively short sequencing time would be sufficient for pathogen identification. They estimated that the turnaround time for MinION 16S sequencing can be reduced to less than eight hours. The study was published in the October 2018 issue of the journal Emerging Infectious Diseases. Image: The MinION is the only portable real-time device for DNA and RNA sequencing (Photo courtesy of Oxford Nanopore Technologies).
Cancer Liquid Biopsy Assay Detects Low-Level Mutations irculating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with variant frequencies that are often low. A cell-free DNA assay has been developed to detect low-frequency mutations in colorectal cancer. The assay initially is to be used for translational studies but a clinical version of the test is under development that might also be applicable to lung and other cancers. An international team of scientists working
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with the Institute of Cancer Research (London, UK; www.icr.ac.uk) has developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction. The team designed a 32-gene assay, 163.3-kilobase pairs in size, using Agilent SureSelectXT HS technology (Agilent Technologies, Santa Clara, CA, USA; www.agilent.com) to which they had early access. The reagent kit incorporates 10-base molecular barcodes. To calculate the sensitivity and specificity of the assay, the team created a
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mixture of cfDNA with 16 known single nucleotide polymorphisms (SNPs) within the targeted region at various concentrations. The group then tested the assay on cfDNA from 28 patients with metastatic colorectal cancer who also had a tumor biopsy sequenced. The cfDNA assay detected 80/91 (88%), of the mutations that had been found by tumor sequencing. The cfDNA sequencing assay also detected mutations in genes that had not previously been analyzed by tumor sequencing. For instance, in four cases that had previously been analyzed via a 5-gene amplicon-based assay, the cfDNA assay detected mutations in the APC gene, a well-known tumor suppressor gene. In addition, the cfDNA assay detected mutations in the FBXW7, CTNNB1, TCF7L2, ATM, and SMAD4 genes. Eleven out of 13 mutations detected resulted in protein changes and that had been reported in the COSMIC database. Marco Gerlinger, MD, a clinical scientist and senior author of the study, said, “We’re interested in discovering resistance mechanisms and new drivers of metastatic disease, so we needed the option of changing around what we want to include in our sequencing assay. While commercially available assays that make use of amplicon technology are customizable, the team also wanted the ability to look at genome-wide copy number alterations, which can be more challenging with amplicon-based assays than with hybrid capturebased ones.” The study was published in the October 2018 issue of the journal Clinical Chemistry. LabMedica International June-July/2019
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