High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and ...quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification.
A novel, strictly anaerobic, short rod-shaped bacterium, designated strain TBC1
, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor ...treating high-strength starch-based organic wastewater. Cells of this strain were 2-4 µm long and 0.4-0.6 µm wide. They were non-motile and Gram-stain-negative. The optimum growth temperature was 30-37 °C, with a range of 20-40 °C. The optimum pH for growth was around pH 7.0, while growth occurred in a range of pH 6.5-9.0. Strain TBC1
grew chemo-organotrophically on a narrow range of carbohydrates under anaerobic conditions. Yeast extract was required for its growth. The major fermentative end products from glucose, supplemented with yeast extract, were acetate, malate, propionate, formate and hydrogen. Doubling time under optimal growth conditions was estimated to be 1 day. The DNA G+C content of strain TBC1
was 49.2 mol% as determined by HPLC. Major cellular fatty acids were C16 : 0, C18 : 0, C16 : 1ω9c and C18 : 1ω9c. Based on its 16S rRNA gene sequence, strain TBC1
was shown to represent a distinct lineage at the family level in the phylum Bacteroidetes. Among previously described species of this phylum, Mucilaginibacter boryungensis BDR-9
(Sphingobacteriaceae) displayed the highest sequence similarity (85.9 %) with strain TBC1
. Phylogenomic analyses using 38-83 single copy marker genes also supported the novelty of strain TBC1
at the family level. Based on its characteristics, strain TBC1
(=JCM 30898
=DSM 100618
) is considered to be the type strain of a novel species of a new genus, Lentimicrobium saccharophilum gen. nov., sp. nov. A new family, Lentimicrobiaceae fam. nov., is also proposed encompassing the strain and related environmental 16S rRNA gene clone sequences.
Abstract
MALDI-TOF MS-based microbial identification relies on reference spectral libraries, which limits the screening of diverse isolates, including uncultured lineages. We present a new strategy ...for broad-spectrum identification of bacterial and archaeal isolates by MALDI-TOF MS using a large-scale database of protein masses predicted from nearly 200,000 publicly available genomes. We verify the ability of the database to identify microorganisms at the species level and below, achieving correct identification for > 90% of measured spectra. We further demonstrate its utility by identifying uncultured strains from mouse feces with metagenomics, allowing the identification of new strains by customizing the database with metagenome-assembled genomes.
Workflows for microbiome community profiling by high-throughput sequencing are prone to sample mix-ups and cross-contamination due to the complexity of the procedures and large number of samples ...typically analyzed in parallel. We employed synthetic 16S rRNA gene spike-in controls to establish a method for tracking of sample identity and detection of cross-contamination in microbiome community profiling assays based on 16S rRNA gene amplicon sequencing (16S-seq). Results demonstrated that combinatorial sample tracking mixes (STMs) can be reliably resolved by Illumina sequencing and faithfully represent their sample of origin. In a single-blinded experiment, addition of STMs at low levels was shown to be sufficient to unambiguously identify and resolve swapped samples. Using artificial admixtures of individually SMT-tagged samples, we further established the ability to detect and quantify cross-contamination down to a level of approximately 1%. The utility of our technique was underscored through detection of an unplanned case of cross-contamination that occurred during this study. By enabling detection of sample mix-ups and cross-contamination throughout 16S-seq workflows, the present technique thus assures provenance of sequence data on a per-sample basis. The method can be readily implemented in standard 16S-seq workflows and its routine application is expected to enhance the reliability of 16S-seq data.
We analyzed the microbial community that developed after 4 years of testing different soil-crop management systems in the savannah–forest transition zone of Eastern Ghana where management systems can ...rapidly alter stored soil carbon as well as soil fertility. The agricultural managements were: (i) the local practice of fallow regrowth of native elephant grass (Pennisetum purpureum) followed by biomass burning before planting maize in the spring, (ii) the same practice but without burning and the maize receiving mineral nitrogen fertilizer, (iii) a winter crop of a legume, pigeon pea (Cajanus cajan), followed by maize, (iv) vegetation free winter period (bare fallow) followed by maize, and (v) unmanaged elephant grass-shrub vegetation. The mean soil organic carbon (SOC) contents of the soils after 4 years were: 1.29, 1.67, 1.54, 0.80 and 1.34%, respectively, differences that should affect resources for the microbial community.
From about 290,000 sequences obtained by pyrosequencing the SSU rRNA gene, canonical correspondence analysis showed that SOC was the most important factor that explained differences in microbial community structure among treatments. This analysis as well as phylogenetic ecological network construction indicated that members of the Acidobacteria GP4 and GP6 were more abundant in soils with relatively high SOC whereas Acidobacteria GP1, GP7, and Actinobacteria were more prevalent in soil with lower SOC. Burning of winter fallow vegetation led to an increase in Bacillales, especially those belonging to spore-forming genera. Of the managements, pigeon-pea cultivation during the winter period promoted a higher microbial diversity and also sequestered more SOC, presumably improving soil structure, fertility, and resiliency.
•50% loss of SOC and biomass in 4 years reshapes the tropical microbial community.•But, soil microbial community structure is remarkably resilient to resource loss.•Acidobacteria groups are most responsive to SOC changes.•Plant residue burning increased the abundance of Bacillales.•N2-fixing fallow crop increased microbial diversity and improved soil fertility.
A novel obligately anaerobic bacterium, designated strain TC1
, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating ...high-strength starch-based wastewater. Cells had a multicellular filamentous morphology, stained Gram-negative and were non-motile. The filaments were flexible, generally >100 μm long and 0.3-0.4 μm wide. Growth of the isolate was observed at 25-43 °C (optimum 37 °C) and pH 6.0-8.5 (optimum pH 7.0). Strain TC1
grew chemo-organotrophically on a range of carbohydrates under anaerobic conditions. Yeast extract was required for growth. The major fermentative end products of glucose, supplemented with yeast extract, were acetate, lactate, succinate, propionate, formate and hydrogen. Co-cultivation with the hydrogenotrophic methanogen
DSM 864
enhanced growth of the isolate. The DNA G+C content was determined experimentally to be 42.1 mol%. The major cellular fatty acids were anteiso-C
, iso-C
and iso-C
3-OH. Based on 16S rRNA gene sequence analysis, strain TC1
belonged to the class
in the phylum
, in which
P3M-1
was its closest phylogenetic relative (88.3 % nucleotide identity). Phylogenomic analyses using 38 and 83 single-copy marker genes also supported the novelty of strain TC1
at least at the genus level. Based on phylogenetic, genomic and phenotypic characteristics, we propose that strain TC1
represents a novel species of a new genus, for which we suggest the name
gen. nov., sp. nov. The type strain of
is strain TC1
( = JCM 30897
= CGMCC 1.5202
).
Droplet microfluidics has emerged as a powerful technology for improving the culturing efficiency of environmental microorganisms. However, its widespread adoption has been limited due to ...considerable technical challenges, especially related to identification and manipulation of individual growth-positive droplets. Here, we combined microfluidic droplet technology with on-chip "fluorescent nucleic acid probe in droplets for bacterial sorting" (FNAP-sort) for recovery of growth-positive droplets and droplet microdispensing to establish an end-to-end workflow for isolation and culturing of environmental microbes. As a proof-of-concept, we demonstrate the ability of our technique to yield high-purity cultures of rare microorganisms from a representative complex environmental microbiome. As our system employs off-the-shelf commercially available equipment, we believe that it can be readily adopted by others and may thus find widespread use toward culturing the high proportion of as-of-yet uncultured microorganisms in different biomes.
By 2012, point of care (POC) testing will constitute roughly one third of the $59 billion in vitro diagnostics market. The ability to carry out multiplexed genetic testing and wireless connectivity ...are emerging as key attributes of future POC devices. In this study, an inexpensive, user-friendly and compact device (termed Gene-Z) is presented for rapid quantitative detection of multiple genetic markers with high sensitivity and specificity. Using a disposable valve-less polymer microfluidic chip containing four arrays of 15 reaction wells each with dehydrated primers for isothermal amplification, the Gene-Z enables simultaneous analysis of four samples, each for multiple genetic markers in parallel, requiring only a single pipetting step per sample for dispensing. To drastically reduce the cost and size of the real-time detector necessary for quantification, loop-mediated isothermal amplification (LAMP) was performed with a high concentration of SYTO-81, a non-inhibiting fluorescent DNA binding dye. The Gene-Z is operated using an iPod Touch, which also receives data and carries out automated analysis and reporting via a WiFi interface. This study presents data pertaining to performance of the device including sensitivity and reproducibility using genomic DNA from Escherichia coli and Staphylococcus aureus. Overall, the Gene-Z represents a significant step toward truly inexpensive and compact tools for POC genetic testing.
Sequencing-based interrogation of gut microbiota is a valuable approach for detecting microbes associated with colorectal cancer (CRC); however, such studies are often confounded by the effect of ...bowel preparation. In this study, we evaluated the viability of identifying CRC-associated mucosal bacteria through centimeter-scale profiling of the microbiota in tumors and adjacent noncancerous tissue from eleven patients who underwent colonic resection without preoperative bowel preparation. High-throughput 16S rRNA gene sequencing revealed that differences between on- and off-tumor microbiota varied considerably among patients. For some patients, phylotypes affiliated with genera previously implicated in colorectal carcinogenesis, as well as genera with less well-understood roles in CRC, were enriched in tumor tissue, whereas for other patients, on- and off-tumor microbiota were very similar. Notably, the enrichment of phylotypes in tumor-associated mucosa was highly localized and no longer apparent even a few centimeters away from the tumor. Through short-term liquid culturing and metagenomics, we further generated more than one-hundred metagenome-assembled genomes, several representing bacteria that were enriched in on-tumor samples. This is one of the first studies to analyze largely unperturbed mucosal microbiota in tissue samples from the resected colons of unprepped CRC patients. Future studies with larger cohorts are expected to clarify the causes and consequences of the observed variability in the emergence of tumor-localized microbiota among patients.
Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This ...study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples.
In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories.
The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract.
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