Microbiomes of the Built Environment A Research Agenda for Indoor Microbiology, Human Health, and Buildings (2017) / Chapter Skim
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Appendix A: An Assessment of Molecular Characterization Tools
Appendix A: An Assessment of Molecular Characterization Tools
Pages 237-276
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From page 237... ...
Nonetheless, molecular manipulation could emerge in the future as an 1 Mention of commercial products or organizations does not imply endorsement by the National Academies of Sciences, Engineering, and Medicine or by members of the Committee on Microbiomes of the Built Environment: From Research to Application.
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From page 238... ...
targets, and shotgun metagenome sequencing. The discussion that follows is focused mainly on the performance of these types of high-throughput technologies in terms of key performance issues, such as specificity, resolution, sensitivity, biologic activities, quantification, and reproducibility, within the context of microbial communities, particularly the microbiomes of the built environment.
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From page 239... ...
Each criterion is discussed in greater detail in the following sections, along with the rationale for the qualitative assessments shown in the table. These qualitative metrics are meant to provide a consensus perspective on microbiome measurement tools through a profile of their strengths and gaps in meeting the goals of recovering microbiome information from built environments.
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Therefore, this table attempts to highlight the major differences among various technologies at a coarse level for general comparison; accuracy may be lost in some cases in this simple table. A hyphen (-)
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APPENDIX A 241 Organism Biologic Functional Toxicologic Viability Activity Coverage Potential Quantification Reproducibility No No Inferred No Relative Middle No No Single gene Yes Relative Middle No No Multi-genes Yes Relative Low No No Single gene No Absolute High No No Multi-genes Yes Absolute High Yes No Single Yes Absolute organism Yes Yes No Yes Absolute No No Peptide Yes Relative Low fragment No No Peptide Yes Absolute High fragment No Yes Metabolites No Relative Low No Yes Metabolites No Absolute High
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Various technologies, such as target gene sequencing, shotgun metagenome sequencing, and gene arrays, are com monly used for detecting specific organisms of interest. The specificity of target gene sequencing is determined primarily by means of the rimersp used for PCR amplification of the target genes.
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. For example, both target sequencing of functional genes and shotgun sequencing of whole communities are capable of providing highly specific information at the level of nucleotide differences on both known and novel genes and pathways (Hess et al., 2011; Mackelprang et al., 2011; Qin et al., 2012; Tringe et al., 2005; Venter et al., 2004)
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and Nanopore technology (Benitez-Paez et al., 2016) to obtain full length of sequencing of phylogenetic markers, which potentially provide more accurate and finer taxonomic resolution of microbial communities and better predict metabolic potentials.
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. For microbial communities of the built environment, and with the use of current sequencing technology, the two practical features for determining informatics success in complete metagenomic assembly are ensuring sufficient biomass to retrieve repre
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Shotgun sequencing typically requires 1 µg DNA for library preparation with sonication for DNA fragmentation without PCR amplification. Various low numbers of PCR amplification cycles (typically 6)
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Rigorous systematic examinations of this issue within the context of the built environment are needed. Despite progress in increasing the accuracy of metagenomic assembly, read binning software tools remain an important complement to improve sensitive organism detection in metagenomic shotgun sequencing since there are fewer restrictions on minimum read depth.
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In the context of the built environment, however, adverse health effects include more than infectious diseases that depend on viability for disease transmission. Evidence suggests that adverse health effects can be caused not only by inhalation of viable airborne pathogens but also by inactive microorganisms and their fragments and component parts (Miller, 1992)
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DNAbased metagenomics provides a "snapshot" of the diversity and functional potential of various microbial taxa with potentially functional populations in a community, but it is not clear whether the populations of these taxa are actively engaged in metabolic activity and reproduction (which is required for a population to persist)
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Although metatranscriptomics is attractive, the challenge is how to obtain sufficiently high quality of total community RNA and to completely remove rRNA prior to sequencing. Obtaining sufficient community RNAs for metranscriptomics could be particularly difficult for microbial communities associated with the built environment, given the challenges of low biomass in samples.
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Metagenomic-based gene annotation gives a preliminary description of biochemical process potential in microbial communities with transcriptionally active pathways identified through RNA-based metagenomic sequencing. Functional gene annotation operates primarily by identifying genes through sequence homology search using translated amino acid query sequences.
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. Such approaches will also benefit from being integrated with molecular networking (Watrous et al., 2012)
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. General extensions from organism interaction networks to gene interaction networks have not been as well developed but could yield new predictive models of important molecular processes required for successful microbial communities (Boon et al., 2014)
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While air quality indices and recommended threshold exposure levels are well defined in terms of certain chemical compounds and particulate matter masses, they are inadequately defined regarding airborne or s urface-borne contaminants of biologic origin. In contrast with the scientific grounding for wastewater and drinking water regulation, bona fide toxicologic characterization of aerosols is only beginning to emerge in the aerosol and built environment community (Brook et al., 2010; Li et al., 2003)
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. There is also little recognition that transmission depends upon host traits as well, which in addition to such factors as age, health condition, and genetic variation in susceptibility, could in the built environment reflect behavioral patterns of space occupancy and use.
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The replication rates are used to infer bacterial growth rates from a single microbiome snapshot and present an important emerging informatics technique for estimating replication rates for microbial communities in the built environment. Because traditional PCR amplification is involved in amplicon-based target sequencing, it appears that target gene sequencing is not quantitative in complex communities as previously demonstrated (Pinto and Raskin, 2012; Tremblay et al., 2015; Zhou et al., 2011)
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. Nevertheless, great caution is necessary in drawing quantitative inferences about microbial community diversity, and in particular absolute abundances, in comparative studies based on amplicon sequencing data.
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Moreover, the potentially dynamic nature of the microbes and the high degree of community complexity mean that natural biologic variation can prevent two labs from producing the same results even after controlling for technical variation. Nevertheless, developing an understanding of the conditions under which accurate and reproducible microbiome measurements in built environments can be made is a foundational requirement for moving investigative research toward practical building design applications that are subject to greater oversight by a broad community of stakeholders.
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Similar challenges also exist for other "omics" technologies, such as proteomics and metabolomics. Although shotgun sequencing avoids many of the biases encountered in amplicon sequencing, the reproducibility problem could be more severe with shotgun sequencing than with amplicon-based target sequencing.
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. Mock communities in the context of human microbiomes helped establish sequencing protocols for sequencing centers, which are working to expand their sequencing capability to support microbial community profiling (Gohl et al., 2016)
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. Studies of indoor microbial communities and the factors that affect them necessarily need to characterize the buildings in which the studies are conducted.
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Applied and Environmental Microbiology 61:3639-3644. Amann, R
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2016. Measurement of bacterial replication rates in microbial communities.
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2011. PCR amplification-independent methods for detection of microbial communities by the high-density microarray phylochip.
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pplied and Environmental Microbiology 73(2)
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Applied and Environmental Microbiology 63(9)
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Applied and Environmental Microbiology 33:1225-1228. HMP (Human Microbiome Project)
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2013. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences.
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Applied and Environmental Microbiology 41:1132-1138. Manor, O., and E
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Applied and Environmental Microbiology 78(16)
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Applied and Environmental Microbiology 67(2)
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Applied and Environmental Microbiology 58:181-186. Suzuki, M
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Applied and Environmental Microbiology 35(2)
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Applied and Environmental Microbiology 77(11)
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2011. Influence of DNA extraction and PCR amplification on studies of soil fungal communities based on amplicon sequencing.
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2013. Random sampling process leads to overestimation of beta-diversity of microbial communities.
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