Considerations for the development and application

Authors: Jim F Huggett Thomas Laver Sasithon Tamisak Gavin Nixon Denise M O’Sullivan Ramnath Elaswarapu David J Studholme Carole A Foy

Publish Date: 2012/12/28

Volume: 18, Issue: 2, Pages: 77-83

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Abstract

Advances in DNA sequencing technology provide the possibility to analyse and characterize the genetic material from microbial populations the microbiome as a whole Such comprehensive analysis of a microbiome using these ‘metagenomic’ approaches offers the potential to understand industrial clinical and environmental microbiology to a level of detail that is unfeasible using conventional molecular or culturebased methods However the complexity offered by metagenomic analysis is also the weakness of this method and poses considerable challenges during analytical standardisation In this manuscript we discuss options for developing control materials for metagenomic analysis and describe our preliminary work investigating how such materials can be used to assist metagenomic measurements The control materials we have developed demonstrate that when performing 16S rDNA sequencing different library preparation methods incorporating adapters before and after the PCR and small primer mismatches can alter the reported metagenomic profile These findings illustrate that metagenomic analysis can be heavily biased by the choice of method and underpin the need for control materials that can provide a useful tool in informing choice of protocol for accurate analysisA metagenome can be defined as the genetic material present within an environmental sample The term environment in this context refers not only to the classic outdoor terrestrial 1 and aquatic 2 3 domains but also environments found within other organisms like the gut 4 5 and associated matrices like probiotic supplements 6 or linked to other manmade scenarios like water purification 7 or during fermentation 8 The field of metagenome analysis metagenomics has grown rapidly as the last 10 years have seen the development and establishment of next generation also termed massively parallel sequencing NGS a technique that has the potential to read over a billion sequences in a single run By combining the power of NGS with microbial population analysis metagenomics offers huge potential to enable us to understand environmental industrial and clinical microbiology By capturing a large proportion of the genetic material the microbiome can be analysed as the complex population of component organisms present in vivo This has the potential to offer a considerable breakthrough when compared to existing molecular and culturebased methods that typically focus on individual species or small groups of organisms There are a number of studies that have already illustrated the power of metagenomics with the Sorcerer II Global Ocean Sampling Expedition 3 and Human Microbiome Project 9 arguably being most prominentMetagenomic analysis does not come without its difficulties the molecular techniques used to generate the data are not only challenging but highly disparate and there are the additional issues associated with how best to manage store and analyse the huge data sets that are produced from a metagenomics experiment 10 Dealing with the informatics challenges is important as they represent a new problem when working with NGS due to the size and complexity of the resultant data This is especially pertinent to metagenomics where sequences of potentially different species of unknown organisms may be being detected and quantified The informatics challenges are not insurmountable though and considerable achievements have been made to facilitate data analysis 11 12 However while dealing with the challenges associated with informatics is fundamental for metagenomic analysis it must not overshadow considerations around the more common issues of technical accuracy and standardization Furthermore the inclusion of control materials be they simpler standards for quality control or more complicated reference materials could provide a valuable tool for understanding and tackling both the technical and the informatics challenges This leads to the question of how such control materials should best support the different approaches used for metagenomics analysisAny standard for metagenomic analysis will need to account for the fact that there are different methods for analysing complex microbial populations following nucleic acid extraction At the simplest dichotomy mixed microbial sequencing analysis can be performed by sequencing either the whole metagenome or a targeted subset of it The simplest approach uses polymerase chain reaction PCR to specifically amplify generic target sequences sequences shared between different microbes like the bacterial 16S ribosomal RNA gene 16S rDNA 13 The PCR products amplicons are then sequenced by an approach termed amplicon sequencing following their manipulation to prepare them for sequencing library preparation Small differences within the amplified deoxyribonucleic acid DNA sequences are identified and compared to databases of known sequences to determine which taxonomic groups are present and with what relative abundance This approach can be very sensitive but is limited to the taxonomic groups defined by the specificity of the PCR primers for example the 16S ribosomal DNA gene is only present within bacteria and so a PCR targeting the sequence will not detect eukaryotic microbes or virusesNext generation sequencing can also be used to perform a broader assessment of the microbial sequences present Applying NGS to whole metagenome sequencing allows abundances of eukaryotic bacterial archaeal microbes as well as viruses to be measured A further level of complexity will also be found by measuring ribonucleic acid RNA as many viruses have RNA genomes rather than DNA RNA may also be a useful proxy for microbial viability Furthermore the types of RNA measured may provide an idea of the metabolic challenges facing an environment and assist in explaining why specific microbes predominate in certain environments 14Whole metagenome sequencing requires fragmenting the extracted genetic material library preparation and sequencing of the library fragments Different NGS instruments provide different data outputs with the current tradeoff being an inverse relationship between length and number of sequences reads The increased number of reads will provide more sequence depth and therefore detect rare sequences while the longer reads will better facilitate the building of the different microbial genomes present within a sample which is useful when dealing with newly discovered microbes for which reference sequences are unavailable

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