Phylogenetic Microarrays

Phylogenetic Microarrays Phylogenetic Microarrays Oleg Paliy, Vijay Shankar and Marketa Sagova-Mareckova 9 Abstract microbial members. Many of these communities Environmental microbial communities are known play pivotal roles in ecosystem processes such as to be highly diverse, often comprising hundreds energy flow, elemental cycling, and biomass pro- and thousands of different species. Such great duction. Energy and nutrients in these systems complexity of these populations, as well as the are processed by intricate networks of metabolic fastidious nature of many of the microorganisms, pathways through multiple community members makes culture-based techniques both inefficient (Duncan et al., 2004; Belenguer et al., 2006; Flint and challenging to study these communities. The et al., 2008; De Vuyst and Leroy, 2011). The sheer analyses of such communities are best accom- complexity of such networks and the difficulty plished by the use of high-throughput molecular involved in culturing the individual members of methods such as phylogenetic microarrays and these communities have challenged researchers next generation sequencing. Phylogenetic micro- who have tried to gain a clearer understanding of arrays have recently become a popular tool for these interactions. Recent advances in molecular the compositional analysis of complex microbial technologies have significantly simplified the communities, owing to their ability to provide analysis of these communities because they simultaneous quantitative measurements of many remove the need to culture and grow community community members. This chapter describes the members individually. Some of the currently currently available phylogenetic microarrays used available molecular techniques include high- in the interrogation of complex microbial commu- throughput sequencing (discussed in chapter 8 of nities, the technology used to construct the arrays, this book), terminal restriction fragment length as well as several key features that distinguish them polymorphism (discussed in Chapter 6), cheq- from other approaches. We also discuss optimiza- uerboard DNA–DNA hybridization, quantitative tion strategies for the development and usage of real-time PCR, fluorescencein situ hybridization, phylogenetic microarrays as well as data analysis and phylogenetic microarrays. Phylogenetic inter- techniques and available options. rogation of small subunit ribosomal RNA (SSU rRNA) molecules using these techniques has led to considerable progress in our understanding of Introduction community structure and dynamics of various Microbes inhabit diverse environments. Some microbial ecosystems (Suau, 2003; Sekirov et al., of these environments include the human intes- 2010). Phylogenetic microarrays, one of the more tinal tract and skin, soil, roots, leaf and bark popular choices among these techniques, have surfaces of plants, ocean waters, deep see vents, been successfully used to quantitatively profile a and air. The ecosystems of such environments variety of microbial communities, including the are populated by communities of microorgan- gastrointestinal tract, sewage sludge, soil, and isms, rather than by individual species, and often air (Brodie et al., 2007; Nemir et al., 2010; Val- contain hundreds and even thousands of different Moraes et al., 2011; Rigsbee et al., 2012). Date: 18:35 Friday 29 November 2013 UNCORRECTED PROOF File: Bioinformatics and Data Analysis 2P 208 | Paliy et al. Although gene expression analysis was the developments in the technology, optimization of original motivation behind the development of usage, applications, and potential future trends in microarrays, their versatility has allowed research- the use of phylogenetic microarrays. ers to adapt this technology for other uses, including phylogenetic analysis. Several types of microarrays have been developed to characterize Current phylogenetic the composition and function of microbial com- microarrays munities, including community genome arrays, The high-throughput and quantitative nature functional gene arrays, and phylogenetic microar- of phylogenetic microarrays makes them an rays. Community genome arrays are constructed excellent solution for researchers who seek to using whole-genomic DNA isolated from species determine the composition of their microbial in pure culture. They allow detection of individual community of interest. Some key features that species and strains in simple and complex com- distinguish different phylogenetic microarrays are munities. Functional gene arrays include probes the choices of phylogenetic markers utilized for to genes encoding important enzymes involved probe design and the experimental platform used in various metabolic processes and are useful for to host these probes (Paliy and Agans, 2012). A monitoring physiological changes in microbial gene or a group of genes that are ubiquitously pre- communities (Waldron et al., 2009; Xie et al., sent among all or at least the majority of species 2010). A good example of a functional gene array of interest often make the best target for phylo- is the GeoChip, which contains tens of thousands genetic analysis. A few already utilized examples of oligonucleotide probes for genes involved in that fit the above criteria include the SSU rRNA biogeochemical cycling of carbon, nitrogen, phos- gene (16S in prokaryotes and 18S in eukaryotes), phorus, and sulfur, for genes involved in metal and the large ribosomal subunit RNA gene (23S and antibiotic resistance, and for genes coding proteins 28S, respectively), genes coding for the heat shock involved in bioremediation of organic compounds proteins GroEL and GroES and for ribosomal (Zhou et al., 2011). Phylogenetic oligonucleotide proteins such as protein S1 (Martens et al., 2007), microarrays (phyloarrays) contain probes com- and in the case of methanogens, the mcrA gene plementary to well conserved and ubiquitous which encodes for methyl coenzyme-M reductase gene sequences (usually the SSU rRNA gene) and (Luton et al., 2002). The SSU rRNA gene is cur- are primarily used for the analysis of microbial rently the most popular choice in part because it community composition and variability (Paliy can be fully and selectively amplified from total and Agans, 2012). Among different array types, genomic DNA with a set of primers complemen- phyloarrays are currently the most popular owing tary to the conserved regions at the beginning and to the availability of a large set of near-full length the end of the gene. Note, however, that the 16S SSU rRNA sequences deposited in NCBI, EMBL, rRNA gene has substantial limitations as a taxo- RDP, and Greengenes databases (see also Chapter nomic marker when attempting to discriminate 7, ‘Repositories of 16S rRNA gene sequences and between closely related taxa, i.e. below the genus taxonomies’). level. This is due to a high level of conservation of The first recognized phylogenetic microarray, this gene sequence across bacterial taxa (Naum et developed by Guschin et al. (1997), was capable al., 2008). As an alternative to rRNA gene, apart of detecting select genera of nitrifying bacteria. from using the genes mentioned above, one can Since then, significant advances have been made also utilize more specific metabolic genes for a with phylogenetic microarrays to improve the particular community of interest. For example, to breadth of detection (total number of different study methanotrophs, methane monooxygenase groups detected), thereby increasing their ver- (pmoA) gene can be used (Bodrossy et al., 2003; satility. Progress has also been made to increase Stralis-Pavese et al., 2011), while nifH gene coding the sensitivity and specificity of phylogenetic for a component of nitrogenase protein complex microarrays (Hazen et al., 2010; Paliy and Agans, can be utilized to profile nitrogen-fixing diazo- 2012). In this chapter, we will discuss the current trophic populations (Zhang et al., 2007). Date: 18:35 Friday 29 November 2013 UNCORRECTED PROOF File: Bioinformatics and Data Analysis 2P Phylogenetic Microarrays | 209 A typical design process for a microarray construction approach allows for a high level of specific to a particular ecosystem or community customization and adaptation. Because no metal usually involves the acquisition of 16S rRNA masks are required, the array design can be updated genes from members of that community (through frequently, and only a limited number of the clone library sequencing, for example) and sub- arrays can be created at any given time. One of the sequent selection of regions within the genes for commercial microarray manufacturers, Agilent, probe design. Region selection can either be done Inc. (USA), uses the process of ink-jet printing manually, based on the availability of unique frag- to print as many as 185,000 features onto a 1 × ments in the hypervariable regions of 16S rRNA 3 inch slide. Recently, microelectrodes have also sequence, or by using mathematical algorithms. been used to construct high-density arrays, where Several software solutions such as ARB, GoArray probes are aligned and concentrated on the array and PhylArray exist to facilitate this process and surface using electrical charges applied to specific provide an optimized automated design of micro- sections of the array. This technique reduces the array probes (Ludwig et al., 2004; Rimour et al., amount of time and labour required for the con- 2005; Militon et al., 2007). Several technologies struction of microarrays. More importantly, due are available for the

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