Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: Wet lab procedure
F. Xavier López‐LabradorJulianne R. BrownNicole FischerHeli HarvalaSander van BoheemenOndřej CinekArzu SayınerTina Vasehus MadsenEeva AuvinenVerena KufnerMichael HuberChristophe RodriguezMarcel JongesMario HönemannPetri SusiHugo SousaPaul E. KlapperAlba Pérez‐CataluñaMarta HernándezRichard MolenkampLia van der HoekRob SchuurmanNatacha CoutoKaroline LeuzingerPeter SimmondsMartin BeerDirk W. HöperSergio KammingaMariet C.W. FeltkampJesús Rodríguez‐DíazEls KeyaertsXiaohui Chen NielsenElisabeth Puchhammer‐StöcklAloys C.M. KroesJavier BuesaJudith BreuerEric C. J. ClaasJutte J.C. de Vries
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Abstract:
Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.Roentgen
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Metagenomic studies have dramatically expanded our knowledge of the microbial world. Furthermore, the amount of sample for sequencing has significantly increased with the development of high-throughput sequencing technologies. However, fully capturing all DNA sequences carried by every microorganism in the environment is still impossible. Therefore, estimating a reasonable and practical amount for sequencing to achieve the objectives is particularly necessary. In the present study, we introduce a novel method for estimating the required minimum amount for metagenomic sequencing for a given goal. We also calculated the genomic proportion of each operational taxonomic unit and the detection efficiency of a specific gene (we have used SSU rRNA gene as an example) based on a given amount for random metagenomic sequencing. The reasonable and practical estimated amount for sequencing in metagenomic studies will provide good reference information when applying high-throughput sequencing for a given goal.
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