Microbial Ecology in the Era of Next Generation Sequencing

Abstract

Sanger sequencing (first generation sequencing) was widely used for molecular biology studies with no or minimum errors. Based on the Sanger sequencing approach, different PCR based methods have been developed to study the microbial communities. Denaturing Gradient Gel Electrophoresis (DGGE) or Terminal Restriction Fragment Length Polymorphism (T-RFLP) has been developed to analyze the uncultured microbial communities in diverse environments [24,25]. These methods allow differentiating gene molecules (in polyacrylamide gels) based on the decreased electrophoresis mobility of a partially melted double stranded DNA molecule at the level of a single base containing a linear gradient of DNA denaturants (a mixture of urea and formamide) or a linear temperature gradient. However, these techniques present several drawbacks as the dominant populations are better revealed and bands from more than one species may be hidden behind a single band resulting in underestimation of bacterial diversity. The same isolate can have different identifiable bands because multiple copies of 16S rRNA gene are present in a single genome of species. The Sanger sequencing based methods present several drawbacks since it is expensive and cannot detect rare microorganisms [26,27]. Next generation sequencing technology (NGS) NGS allows massively parallel sequencing with thousands to millions of sequences in one experiment at considerably low cost compared to Sanger method. There are no requirements of bacterial cloning of DNA fragments and electrophoresis, since NGS libraries preparation process are carried out in a cell-free system and the sequence output is directly detected. However, it produces shorter reads with a higher error rate than Sanger sequencing [28-30]. Moreover, secondary structure and thermal stability of the genome affects the efficiency of PCR amplification of the genomic fragments. There are different NGS platforms (e.g. Illumina, Pacific Biosciences, Ion Torrent, SOLiD) with different principle and divergent features such as run times, yields and read lengths [31] (Table 1). Advances in NGS have revolutionized the field of microbial ecology. Any NGS study follow some common steps such as sample and metadata collection, DNA extraction, library construction, sequencing and read preprocessing followed by quantitative analysis and functional binning [32,33]. Collection of environment sample is the first step in any metagenomic study. But, the problem faced in microbial ecology studies are that no information is available about the amount of required