PyroTRF-ID: A Bioinformatics Methodology for Profiling Microbiomes with T-RLFP and Amplicon Sequencing Data

Microbial ecologyMicrobial ecology benefits from the gradual developmentsDevelopment in bioanalyticsBioanalytics to profileProfile microbiomesMicrobiome at increasing resolutionsResolution. Massive sequencingSequencing is gradually replacing classical fingerprintingFingerprinting techniques. Terminal-restriction fragment lengthLength polymorphism (T-RFLPT-RFLP) has been frequently used to characterize the compositionsComposition of microbial communitiesMicrobial community, in combination with cloningCloning-sequencingCloning-sequencing to associate operational taxonomic unitsOperational taxonomic unit to populationsPopulation. Amplicon sequencingAmplicon sequencing offers high resolutionResolution in communityCommunity profilingProfiling and directly identifies phylotypesPhylotype together with their relative abundanceRelative abundance. In the transition from T-RFLPT-RFLP to amplicon sequencingAmplicon sequencing, the PyroTRF-IDPyroTRF-ID bioinformaticsBioinformatics methodologyMethodology was developed here to use pyrosequencingPyrosequencing datasetsDataset for identifying terminal restriction fragmentsTerminal restriction fragment (T-RFs) in the numerous experimental T-RFLPT-RFLP profilesExperimental T-RFLP profile. T-RFLP was used in this research as a rapid routine analysisRoutine analyses to study microbial selectionMicrobial selection mechanisms in granular sludgeGranular sludge. PyroTRF-IDPyroTRF-ID generates digital T-RFLP profilesDigital T-RFLP profile from 16S rRNARRNA geneGene amplicon sequencingAmplicon sequencing datasetsDataset, and match them to experimental ones for associating T-RFs to bacterial taxaTaxa. The workflowWorkflow was developed and tested with dataData collected from both granular sludgeGranular sludge and groundwaterGroundwater environmentsEnvironment, highlighting the applicability of the procedureProcedure to any microbial communityMicrobial community from engineered to more complex natural environmentsNatural environment. On average, 63 ± 18% of all experimental T-RFs were affiliated to phylotypesPhylotype, i.e., considerably enhancing taxonomic affiliationsTaxonomic affiliation when compared to cloningCloning-sequencingCloning-sequencing. While current incentives aim at going all for sequencingSequencing, PyroTRF-IDPyroTRF-ID associates the fast throughputFast throughput of T-RFLPT-RFLP and the high resolutionResolution of amplicon sequencingAmplicon sequencing to optimize analytical and computational resourcesComputational resource in the profilingProfiling of microbial communitiesMicrobial community. PyroTRF-IDPyroTRF-ID can be adapted to any sequencingSequencing technologyTechnology. Amplicon sequencingAmplicon sequencing can be used on selected reference samples to identify phylotypesPhylotype, and T-RFLPT-RFLP for cheap routine analysesRoutine analyses at process lineProcess line to deliver rapid informationInformation for process managementProcess management.