Advances in high-throughput sequencing (HTS) have fostered rapid developments in the field of microbiome research, and massive microbiome datasets are now being generated. However, the diversity of software tools and the complexity of analysis pipelines make it difficult to access this field. Here, we systematically summarize the advantages and limitations of microbiome methods. Then, we recommend specific pipelines for amplicon and metagenomic analyses, and describe commonly-used software and databases, to help researchers select the appropriate tools. Furthermore, we introduce statistical and visualization methods suitable for microbiome analysis, including alpha- and beta-diversity, taxonomic composition, difference comparisons, correlation, networks, machine learning, evolution, source tracing, and common visualization styles to help researchers make informed choices. Finally, a step-by-step reproducible analysis guide is introduced. We hope this review will allow researchers to carry out data analysis more effectively and to quickly select the appropriate tools in order to efficiently mine the biological significance behind the data.

/pdf/a-practical-guide-to-amplicon-and-metagenomic-analysis-of-3b4dhcga92.pdf

A practical guide to amplicon and metagenomic analysis of microbiome data

Although considerable progress has been made in recent years regarding the classification of bacteria assigned to the phylum Bacteroidetes, there remains a need to further clarify taxonomic relationships within a diverse assemblage that includes organisms of clinical, piscicultural, and ecological importance. Bacteroidetes classification has proved to be difficult, not least when taxonomic decisions rested heavily on interpretation of poorly resolved 16S rRNA gene trees and a limited number of phenotypic features. Here, draft genome sequences of a greatly enlarged collection of genomes of more than 1,000 Bacteroidetes and outgroup type strains were used to infer phylogenetic trees from genome-scale data using the principles drawn from phylogenetic systematics. The majority of taxa were found to be monophyletic but several orders, families and genera, including taxa proposed long ago such as Bacteroides, Cytophaga, and Flavobacterium but also quite recent taxa, as well as a few species were shown to be in need of revision. According proposals are made for the recognition of new orders, families and genera, as well as the transfer of a variety of species to other genera. In addition, emended descriptions are given for many species mainly involving information on DNA G+C content and (approximate) genome size, both of which can be considered valuable taxonomic markers. We detected many incongruities when comparing the results of the present study with existing classifications, which appear to be caused by insufficiently resolved 16S rRNA gene trees or incomplete taxon sampling. The few significant incongruities found between 16S rRNA gene and whole genome trees underline the pitfalls inherent in phylogenies based upon single gene sequences and the impediment in using ordinary bootstrapping in phylogenomic studies, particularly when combined with too narrow gene selections. While a significant degree of phylogenetic conservation was detected in all phenotypic characters investigated, the overall fit to the tree varied considerably, which is one of the probable causes of misclassifications in the past, much like the use of plesiomorphic character states as diagnostic features.

/pdf/analysis-of-1-000-type-strain-genomes-improves-taxonomic-345doatvi9.pdf

Analysis of 1,000 Type-Strain Genomes Improves Taxonomic Classification of Bacteroidetes .

Microbial communities are essential components of aquatic ecosystems through their contribution to food web dynamics and biogeochemical processes. Aquatic microbial diversity is immense and a general challenge is to understand how metabolism and interactions of single organisms shape microbial community dynamics and ecosystem-scale biogeochemical transformations. Metagenomic approaches have developed rapidly, and proven to be powerful in linking microbial community dynamics to biogeochemical processes. In this review, we provide an overview of metagenomic approaches, followed by a discussion on some recent insights they have provided, including those in this special issue. These include the discovery of new taxa and metabolisms in aquatic microbiomes, insights into community assembly and functional ecology as well as evolutionary processes shaping microbial genomes and microbiomes, and the influence of human activities on aquatic microbiomes. Given that metagenomics can now be considered a mature technology where data generation and descriptive analyses are relatively routine and informative, we then discuss metagenomic-enabled research avenues to further link microbial dynamics to biogeochemical processes. These include the integration of metagenomics into well-designed ecological experiments, the use of metagenomics to inform and validate metabolic and biogeochemical models, and the pressing need for ecologically relevant model organisms and simple microbial systems to better interpret the taxonomic and functional information integrated in metagenomes. These research avenues will contribute to a more mechanistic and predictive understanding of links between microbial dynamics and biogeochemical cycles. Owing to rapid climate change and human impacts on aquatic ecosystems, the urgency of such an understanding has never been greater.

/pdf/linking-metagenomics-to-aquatic-microbial-ecology-and-1st6q2tcsw.pdf

Linking metagenomics to aquatic microbial ecology and biogeochemical cycles

New insights into the effect of sludge proteins on the hydrophilic/hydrophobic properties that improve sludge dewaterability during anaerobic digestion.

Mangroves, as a blue carbon reservoir, provide an environment for a variety of microorganisms. Mangroves lie in special locations connecting coastal and estuarine areas and experience fluctuating conditions, which are expected to intensify with climate change, creating a need to better understand the relative roles of stochastic and deterministic processes in shaping microbial community assembly. Here, a study of microbial communities inhabiting mangrove sediments across southeastern China, spanning mangroves in six nature reserves, was conducted. We performed high-throughput DNA sequencing of these samples and compared them with data of 1,370 sediment samples collected from the Earth Microbiome Project (EMP) to compare the microbial diversity of mangroves with that of other biomes. Our results showed that prokaryotic alpha diversity in mangroves was significantly higher than that in other biomes and that microbial beta diversity generally clustered according to biome types. The core operational taxonomic units (OTUs) in mangroves were mostly assigned to Gammaproteobacteria, Deltaproteobacteria, Chloroflexi, and Euryarchaeota The majority of beta nearest-taxon index values were higher than 2, indicating that community assembly in mangroves was better explained through a deterministic process than through a stochastic process. Mean annual precipitation (MAP) and total organic carbon (TOC) were main deterministic factors explaining variation in the microbial community. This study fills a gap in addressing the unique microbial diversity of mangrove ecosystems and their microbial community assembly mechanisms.IMPORTANCE Understanding the underlying mechanisms of microbial community assembly patterns is a vital issue in microbial ecology. Mangroves, as an important and special ecosystem, provide a unique environment for examining the relative importance of stochastic and deterministic processes. We made the first global-scale comparison and found that microbial diversity was significantly different in mangrove sediments compared to that of other biomes. Furthermore, our results suggest that a deterministic process is more important in shaping microbial community assembly in mangroves.

https://msystems.asm.org/content/msys/4/5/e00442-19.full.pdf

Prokaryotic Diversity in Mangrove Sediments across Southeastern China Fundamentally Differs from That in Other Biomes.

The pure culture of prokaryotes remains essential to elucidating the role of these organisms. Scientists have reasoned that hard to cultivate microorganisms might grow in pure culture if provided with the chemical components of their natural environment. However, most microbial species in the biosphere that would otherwise be “culturable” may fail to grow because of their growth state in nature, such as dormancy. That means even if scientist would provide microorganisms with the natural environment, such dormant microorganisms probably still remain in a dormant state. We constructed an enrichment culture system for high-efficiency isolation of uncultured strains from marine sediment. Degree of enrichment analysis, dormant and active taxa calculation, viable but non-culturable bacteria resuscitation analysis, combined with metatranscriptomic and comparative genomic analyses of the interactions between microbial communications during enrichment culture showed that the so-called enrichment method could culture the “uncultured” not only through enriching the abundance of “uncultured,” but also through the resuscitation mechanism. In addition, the enrichment culture was a complicated mixed culture system, which contains the competition, cooperation, or coordination among bacterial communities, compared with pure cultures. Considering that cultivation techniques must evolve further—from axenic to mixed cultures—for us to fully understand the microbial world, we should redevelop an understanding of the classic enrichment culture method. Enrichment culture methods can be developed and used to construct a model for analyzing mixed cultures and exploring microbial dark matter. This study provides a new train of thought to mining marine microbial dark matter based on mixed cultures.

/pdf/metatranscriptomic-and-comparative-genomic-insights-into-46miurvu9m.pdf

Metatranscriptomic and comparative genomic insights into resuscitation mechanisms during enrichment culturing.

A novel Gram-stain-negative, strictly aerobic, non-flagellated and rod-shaped bacterium, designated HF004T, was isolated from a marine sediment sample collected from the coast of Weihai, China The strain grew optimally at 28 °C, pH 75–80 and in the presence of 20–30 % (w/v) NaCl Based on the 16S rRNA gene sequence analysis, strain HF004T was a member of the genus 
Halioglobus
, appearing to be closely related to 
Halioglobus
pacificus (961 %) and 
Halioglobus japonicus
(956 %) The major fatty acids were summed feature 3 (ie C16 : 1ω7c and/or iso-C15 : 0 2-OH), C17 : 1ω8c and C18 : 1ω7c The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol The predominant respiratory quinone was Q-8 The DNA G+C content was 572 mol% Cells of strain HF004T were rod-shaped and formed circular, mucous and beige-pigmented colonies on marine agar after incubation for 72 h at 28 °C On the basis of phenotypic, genotypic and phylogenetic evidence, strain HF004T is presented as a novel species, for which the name Halioglobus lutimaris sp nov is proposed The type strain is HF004T (=KCTC 42395T=MCCC 1H00127T)

Halioglobus lutimaris sp. nov., isolated from coastal sediment

A Gram-stain-negative, strictly aerobic, non-flagellated, yellow-pigmented, rod-shaped bacterial strain, designated 0182T, was isolated from Gracilaria blodgettii, an algae of phylum Rhodophyta collected from coast of Lingshui county, Hainan, China (110° 03′ 44.2′′ E, 18° 24′ 29.8′′ N). The strain grew optimally at 30 °C, pH 7.0–7.5 and in the presence of 2.0–3.0 % (w/v) NaCl. Cells of strain 0182T were approximately 0.9–2.5 µm in length and 0.2–0.4 µm in width. The major respiratory quinone was MK-7. The predominant cellular fatty acids were iso-C15 : 0, C15 : 0 and iso-C17 : 0 3-OH. The major polar lipids were phosphoaminolipid, glycolipid, two unknown aminolipids and four unknown lipids. The DNA G+C content was approximately 35.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 0182T was phylogenetically related to members of the genus 
Crocinitomix
and was closely related to 
Crocinitomix catalasitica
with 94.6 % sequence similarity. On the basis of genotypic and phenotypic characteristics and phylogenetic evidence, strain 0182T is thought to represent a novel species of the genus 
Crocinitomix
, for which the name Crocinitomix algicola sp. nov. is proposed. The type strain is 0182T (=KCTC 42868T =MCCC 1H00128T).

Crocinitomix algicola sp nov., isolated from Gracilaria blodgettii

A novel Gram-stain-negative, strictly aerobic, rod-shaped and agar-hydrolysing bacterium, designated D2T, was isolated from a marine sediment sample collected from the coast of Weihai, China (37° 31′ 59″ N 122° 03′ 47″ E). The cells were motile by a lateral flagellum. Growth was observed at 10–42 °C, at pH 6.0–9.0 and with 0.5–8 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain D2T belonged to the genus 
Catenovulum
, appearing closely related to 
Catenovulum agarivorans
YM01T (96.3 % 16S rRNA gene sequence similarity) and 
Catenovulum maritimum
Q1T (93.9 %). The dominant fatty acids were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), C16 : 0 and C10 : 0 3-OH. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, two phosphoaminolipids, two unknown lipids and three phospholipids. Ubiquinone 8 (Q-8) was found to be the major respiratory quinone. The DNA G+C content was 40.4 mol%. On the basis of genotypic, phenotypic and phylogenetic evidence, strain D2T is presented as a representative of a novel species of the genus 
Catenovulum
, for which the name Catenovulum sediminis sp. nov. is proposed. The type strain is D2T (=KCTC 42869T=MCCC 1H00129T). An emended description of the genus 
Catenovulum
is also provided.

Catenovulum sediminis sp. nov., isolated from coastal sediment

A Gram-negative, facultatively anaerobic, non-motile, rod-shaped and orange-coloured bacterium, designated N62T, was isolated from marine sediment of the coast of Weihai, PR China. Strain N62T was found to grow optimally at 28–30 °C, pH 7.0–7.5 and with 2.0–3.0 % (w/v) NaCl. The dominant cellular fatty acids of strain N62T were iso-C15 : 0, iso-C15 : 0 G, iso-C17 : 0 3-OH and iso-C17 : 1 3-OH. The major respiratory quinone was MK-6, and the DNA G+C content was 35.3 mol%. The predominant polar lipids were phosphatidylethanolamine, two unidentified aminolipids, an unidentified glycolipid and three unidentified lipids. Phylogenetic analysis based on 16S rRNA gene sequences revealed that N62T was a member of the family 
Crocinitomicaceae
and had a 16S rRNA gene sequence similarity of 95.8–97.2 % with recognized 
Brumimicrobium
species. On the basis of the phylogenetic and phenotypic evidences, strain N62T represents a novel species of the genus 
Brumimicrobium
, for which the name Brumimicrobium aurantiacum sp. nov. is proposed. The type strain is N62T (=KCTC 42589T=MCCC 1H00117T).

Ming-Jing Shi

Papers

Metatranscriptomic and comparative genomic insights into resuscitation mechanisms during enrichment culturing.

Halioglobus lutimaris sp. nov., isolated from coastal sediment

Crocinitomix algicola sp nov., isolated from Gracilaria blodgettii

Catenovulum sediminis sp. nov., isolated from coastal sediment

Brumimicrobium aurantiacum sp. nov., isolated from coastal sediment