The BES Microbial Ecology Special Interest Group is running a workshop today (Thursday 2 November) on Novel Tools for Microbial Ecology. To compliment this workshop, Xavier Harrison has edited a Virtual Issue of the best Methods in Ecology and Evolution articles on advances in methods of studying microbial evolution and ecology from the past few years.

Advances in Next-Generation Sequencing (NGS) technology now allow us to study associations between hosts and their microbial communities in unprecedented detail. However, studies investigating host-microbe interactions in the field of ecology and evolution are dominated by 16S and ITS amplicon sequencing. While amplicon sequencing is a useful tool for describing microbial community composition, it is limited in its ability to quantify the function(s) performed by members of those communities. Characterising function is vital to understanding how microbes and their hosts interact, and consequently whether those interactions are adaptive for, or detrimental to, the host. The articles in this Virtual Issue cover a broad suite of approaches that allow us to study host-microbe and microbe-microbe interactions in novel ways.

All of the articles in the Microbial Methods Virtual Issue will be freely available for the next two months. You can find out a little more about each one below.

Ecological Interactions and Microbial Communities

We begin with Suzuki et al.’s article ‘An equation-free method reveals the ecological interaction networks within complex microbial ecosystems‘. Mapping the network of ecological interactions is a key to understanding the composition, stability, function and dynamics of microbial communities. The authors developed the sparse S-map method (SSM), which generates a sparse interaction network from a multivariate ecological time series without presuming any mathematical formulation for the underlying microbial processes. It provides a powerful framework to infer ecological interaction networks of microbial communities in many environments.

Sequence-Based Identification: A Field Guide

Creer et al. provide an excellent description of the the different technologies available within the omics toolbox in their article ‘The ecologist’s field guide to sequence-based identification of biodiversity‘. The article covers both field and lab methods and will take you through sampling and preservation of genomic material, nucleic acid extraction approaches, marker gene assessment, metatranscriptomics,  single-cell genomics and more. The authors also discussed their article in a Methods in Ecology and Evolution podcast earlier this year.

Flow Cytometry

Translating raw flow cytometry data (top panels) into diversity estimates (bottom panels) for two surveys of a freshwater microbial ecosystem (
Translating raw flow cytometry data (top panels) into diversity estimates (bottom panels) for two surveys of a freshwater microbial ecosystem (

Measuring microbial diversity is important for ecosystem characterisation, ecosystem monitoring and hypothesis testing. In ‘Measuring the biodiversity of microbial communities by flow cytometry‘ Props et al. demonstrate that sensitive single-cell measurements of phenotypic attributes, obtained via flow cytometry, can provide fast (within minutes!) first-line assessments of microbial diversity dynamics. This fast, robust analysis method for monitoring microbial biodiversity has applied and fundamental implications. You can find out more about this paper in Exploring Microbial Diversity: From the Sequence to the Cell.

Extracting DNA

Sundberg et al.’s new method makes routine amplification of DNA from single microscopic fungal thalli possible, even from small species or poorly developed individuals. In ‘A crush on small fungi: An efficient and quick method for obtaining DNA from minute ascomycetes‘ they explain how the approach has eliminated the need for additional treatments like laborious freeze-thaw cycles, enzymes, or lysing agents. As all researchers working with minute uncultivable organisms in many respects face the same problems, the methodology described here has a potential to be widely applicable.

Conserved Genomic Elements

In ‘Identifying conserved genomic elements and designing universal bait sets to enrich them‘ Brian Faircloth describes a universal workflow for identifying conserved genomic regions in available genomic data and for designing targeted enrichment baits to collect data from these conserved regions. Targeted enrichment of conserved genomic regions is a popular method for collecting large amounts of sequence data from non-model taxa for phylogenetic, phylogeographic and population genetic studies. This software workflow will allow you to identify thousands of conserved loci and design sequence capture baits to target them.

Detection of Parasitoid and Pathogen DNA

Molecular methods are facing increasing application on a broad remit in ecological studies, including work on parasites, parasitoids and pathogens. To optimise the application of molecular diagnostics in situations where infection or parasitism rates are low, Sint et al. developed a pooling approach that is applicable to any host–pathogen and host–parasite/parasitoid system. In ‘A two-dimensional pooling approach towards efficient detection of parasitoid and pathogen DNA at low infestation rates‘ the authors show that their approach presents an efficient and sensitive means for screening of large sample numbers at low parasitism and infection rates.

DNA Barcoding and Metabarcoding

In ‘Quantifying uncertainty of taxonomic placement in DNA barcoding and metabarcoding‘ Somervuo et al. evaluate the performance of the recently proposed probabilistic taxonomic placement method PROTAX by applying it to both annotated reference sequence data as well as to unknown environmental data. They conclude that reliable species-level identification from environmental samples is still challenging and that neglecting identification uncertainty can lead to spurious inference. This article was part of the Technological Advances at the Interface Between Ecology and Statistics Special Feature.

Soil Microbial Communities and Heavy Metal Concentration

Heavy metals in soils can affect the growth of soil microbial communities

Accumulations of heavy metals in soils can affect the growth of soil microbial communities, but this depends on the capability of micro-organisms to tolerate heavy metals. In ‘Identification of response classes from heavy metal-tolerant soil microbial communities by highly resolved concentration-dependent screenings in a microfluidic system‘ Kürsten et al. present a new methodological approach for the assessment of the growth–response behaviour of soil microbial communities in response to increasing heavy metal concentration (copper) using the droplet-based micro-segmented flow technique.

Printing Soil

Traditional high throughput methods for isolating microorganisms require pre-processing steps to remove the living species from their solid-phase microniche, creating a liquid-phase sample. This process destroys near-neighbor relationships that could be crucial to culturing and studying the microorganisms to be isolated. Ringeisen et al. present an automated, high throughput method is described here that isolates pure microbial cultures and spatially related microbial consortia directly from a solid-phase complex environmental sample in ‘Printing soil: a single-step, high-throughput method to isolate micro-organisms and near-neighbour microbial consortia from a complex environmental sample‘.


Oligotyping is a novel supervised computational method that allows researchers to investigate the diversity of closely related but distinct bacterial organisms. Eren et al. describe this method in their 2013 article ‘Oligotyping: differentiating between closely related microbial taxa using 16S rRNA gene data‘. The process can resolve the distribution of closely related organisms across environments and unveil previously overlooked ecological patterns for microbial communities.

Modelling Microbial Populations and Communities

Simple interactions between viruses (bacteriophages) and bacteria can be included in microPop.

The most recent article is this Virtual Issue, ‘microPop: Modelling microbial populations and communities in R‘ by Kettle et al. introduces the R packeage microPop – a dynamic model based on a functional representation of different microbiota. It simulates the dynamics and interactions of microbial populations by solving a system of ordinary differential equations that are constructed automatically based on a description of the system.

Fungal Internal Transcribed Spacer Sequences

Studying fungal biodiversity using data generated from Illumina MiSeq sequencing platforms poses a number of bioinformatic challenges. To help overcome these, Gweon et al. introduce PIPITS in ‘PIPITS: an automated pipeline for analyses of fungal internal transcribed spacer sequences from the Illumina sequencing platform‘. PIPITS is open-source stand-alone suite of software for automated processing of Illumina MiSeq sequences for fungal community analysis.

Experimental Evolution

Experimental evolution is an important research framework for evolutionary biologists as it allows direct testing of fundamental theories about adaptation and diversity, which often requires the tracking of genotypes or alleles over time. Koch et al.’s novel approach of combining multiplex droplet digital polymerase chain reaction with experimental evolution for tracking genotype frequencies in different experimental populations over time is explained in ‘Use of ddPCR in experimental evolution studies‘. This method is precise and accurate for detecting and quantifying targets of interest, especially as compared to traditional PCR methods.

DNA Melting Analysis

How valuable is DNA melt peak analysis for the identification of pests and pathogens significant to biosecurity? In ‘Evaluation of DNA melting analysis as a tool for species identification‘ Winder et al. show that it is a useful and robust tool for the rapid detection of PCR products and the identification of organisms.They also show that it is quicker and cheaper than PCR or AGE.

Microbial Multilocus Sequence Typing

In ‘MLST@SNaP: user-friendly software for simplification of multilocus sequence typing and dissemination of microbial population analyses‘ Soares and Araujo describe mlst@snap, a fast, user-friendly and automated process to convert thousands of MLST sequences or sequence types into SNaP profiles and vice versa. The software largely increases the promptness of microbial population data analyses, being presently designed for the micro-organisms. It’s freely available and is accompanied by tutorial, example files and some data.

Gut Microbiomes and Host Health

Gut microbiome studies in wildlife species help us to understand the effects of intrinsic and extrinsic factors on the variation of bacterial communities, and the health of the host organism. Menke et al. show that preliminary tests are needed to assess up to which point bacterial data derived from faecal samples can still deliver reliable results in ‘Shifts in the gut microbiome observed in wildlife faecal samples exposed to natural weather conditions: lessons from time-series analyses using next-generation sequencing for application in field studies‘.

Environmental Sequencing Data

The nuclear ribosomal internal transcribed spacer (ITS) region is the primary choice for molecular identification of fungi. Extracting and identifying its two highly variable spacers from large taxonomic and environmental data sets is often difficult though, and many ITS sequences are incorrectly delimited in the public sequence databases. In the final article of this Virtual Issue – ‘Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data‘ – Bengtsson-Palme et al. introduce ITSx. ITSx is a Perl-based software tool to extract both variable spacers, the conserved 5.8S gene and full-length ITS sequences.

We hope that you find all of the articles in the Microbial Methods Virtual Issue interesting, useful and enjoyable. They will be free until the end of 2017.