A Handbook of Microbial Genomics: The Past, Present and Future

1. A Handbook of Microbial Genomics: The Past, Present and Future Over the past decade, advances in nucleic acid sequencing and mass spectrometry have enabled faster and more informative analysis of metagenomic, transcriptome, proteomics and metabolomics. Microbes began to appear on Earth about 3.5 billion years ago. They eventually occupied every position in the Earth's biosphere. Although microbes are known to be responsible for many of the key functions on Earth, such as carbon and nutrient cycling, and determining the health and disease of the earth's plants and animals, 99% of microbes are still currently considered to be undiscovered. In addition, research on specific functions of the microbial population become very complex due to the complicated microbial population diversity. Fortunately, technological advances over the past few decades have greatly facilitated the study of complex microorganisms and their functions. Nucleic acid sequencing The phenomenal growth in the speed and throughput of nucleic acid sequencing technology has facilitated advances in microbial research. In particular, Next Generation sequencing has revolutionized the traditional Sanger sequencing method that has dominated the past three decades (1977-2005). The first bacterial genome that was completely sequenced using the Sanger method was Haemophilus influenza in 1995 (E. coli was completed in 1997). At present, bacterial genome sequencing can be completed in a few hours due to the fast and inexpensive NextGen sequencing platform available.

2. Single molecule sequencing Emerging sequencing technologies, for example, single-molecule-based DNA sequencers can complete microbial genome research faster. One example is the single molecule real time (SMRT) technique from PacBio, which relies on a trapped DNA polymerase and a zero mode waveguide to direct light energy through a small amount of liquid. PacBio currently offers DNA sequence reads of 10-25 kbp in length and ~300 Mbp per SMRT chamber (http://www.pacb.com). Due to the use of binding polymerases, the PacBio platform can detect abnormalities due to polymerase swings, allowing direct reading of DNA modifications. The Oxford Nanopore sequencing platform is another emerging and promising single molecule sequencer. Unlike current platforms, the Oxford platform does not rely on synthetic sequencing, but sorts nucleic acid molecules directly through Nanopore. Oxford Nanopore can detect DNA modifications like PacBio platform with an average read length of ~1-2 kbp and the longest read length provided by any sequencer (> 90 kbp). The main advantage of the Oxford Nanopore sequencer is its thumb size, which can be analyzed in real-time using wireless technology on a personal laboratory computer. Over the past five years, the PacBio platform has become a powerful technology for sequencing microbial samples for de novo and metagenomics assembly. It is worth noting that the PacBio platform requires a small amount of high molecular weight DNA (> 40 kbp) for library preparation, which limits the sample ranges that can be sequenced. Oxford Nanopore provides an inexpensive way to potentially sequence large fragments (> 50 kbp). However, the main challenge of these new technologies is to obtain high quality and large molecular weight DNA of hundreds of kilobases or even megabases in length. The Oxford and PacBio platforms are still too low throughput for large-scale studies, but for sequence assembly applications, these single-molecule sequences provide future prospects for microbial communities. Sequencing complex microbial communities Improvements in DNA sequencing technology have enabled people to discover many microbial components and potential functions of our bodies. Most of the information about microbes comes from the 16S rRNA gene sequenced by NextGen as a phylogenetic marker for bacteria and archaea. In addition, NextGen sequencing of total DNA (ie, genomics) has enabled the identification of functional genes associated with specific microbial populations in different environments. For example, some studies have defined functional gene composition in water and sediment samples after deep water horizon oil spills in the Gulf of Mexico and thawed permafrost. However, most of these genes have no known function, reflecting the vast diversity and biochemical potential of environmental microorganisms yet to be discovered. Metatranomics

3. Metatranomics sequences total mRNA and reveals which genes are expressed by specific organisms on spatial and temporal scales. Metatranomics provides knowledge of the expression of microbial genes in a variety of ecosystems, including acid mine drainage, intestines, oceans and soils. For example, Gilbert et al. used a macro transcriptome to determine the seasonal expression pattern of the microbiota in the English Channel. Recently, our researchers used Metatranomics to infer which organism is active in the soil. Although Verrucomicrobia is very abundant in the soil being investigated, very few mRNA transcripts map to its genome, indicating that they are actually transcriptionally quiescent. In contrast, the Firmicutes genome was found to be transcriptionally active. This reveals the utility of Metatranomics in validating metagenomics and understanding the relative activities of different members of the microbial community. Proteomic and metabolomic measurement based on mass spectrometry Although advances in DNA sequencing have enabled a better understanding of microbial phylogeny and functional gene composition in microbes, it is also desirable to understand which proteins (ie, macroproteomics) and metabolites (ie, metabolomics) are produced under specific conditions. And how disturbances affect microbial function are waiting to be uncovered. The measurement of proteins and metabolites produced by microorganisms in different samples is mainly achieved by mass spectrometry (MS).