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Omics Approach to Understanding Microbial Diversity
Published in Jyoti Ranjan Rout, Rout George Kerry, Abinash Dutta, Biotechnological Advances for Microbiology, Molecular Biology, and Nanotechnology, 2022
Shilpee Pal, Arijit Jana, Keshab Chandra Mondal, Suman Kumar Halder
On the other hand, shotgun sequencing includes the entire genomic content of individuals without selecting any particular gene (Scholz et al., 2016). Thus, it can be used to analyze the compositional and functional potential of microbial communities as well as taxonomic profiling. A typical shotgun metagenomics approach is comprised of five steps: (1) collection, processing, and sequencing of samples; (2) preprocessing of sequencing reads; (3) analysis of sequences for profiling the genomic, taxonomic, and functional features of the microbiome; (4) postprocessing analysis through statistics and biology; and (5) validation of the result. Several computational approaches are available to perform each step (Table 2.1). Shotgun metagenomics is becoming popular in biomedical and environmental applications, as well as to identify extremophiles and symbiotic interactions between microbes and hosts (Cowan et al., 2015; Belda-Ferre et al., 2012; Woyke et al., 2006). Shotgun sequencing can provide higher taxonomic information than targeted metagenomics. It can also identify many uncharacterized species, especially those present in low abundance.
Bioinformatics and Genomics Breakthroughs That Enabled the Microbiome Revolution
Published in Kenneth Wunch, Marko Stipaničev, Max Frenzel, Microbial Bioinformatics in the Oil and Gas Industry, 2021
The alternative or in many cases, complementary, approach is shotgun sequencing, either of genomic DNA (metagenomics) or mRNA (metatranscriptomics). Shotgun sequencing has the theoretical advantage of capturing everything and can resolve taxa to species and even to strains. It is not efficient, requiring millions of sequences per sample, but as sequencing costs have fallen, so have costs per sample. Metagenomics or metatranscriptomics data are less simple to process, but several packages have been released to convert these data into microbial profiles. These data also hold information about the metabolic pathways that were either encoded by the microbes (metagenomics) or expressed by the microbes (metatranscriptomics). The ability to directly measure expressed pathways has been cited as an advantage for metatranscriptomics over metagenomics. However, since the most abundant microbes dominate the signals in both and the most highly expressed pathways dominate for metatranscriptomics, it is not clear that the theoretical advantage translates into a practical one unless one sequences more deeply than in most studies (reviewed in Migun, Lo and Chain 2019). In practice, metagenomic shotgun sequencing is often used on a subset of samples after amplicon sequencing. While this approach has advantages in cost, the two approaches can best be considered as having different biases as will be explored in more detail in the following section on data analysis.
Principles and Techniques for Deoxyribonucleic Acid (DNA) Manipulation
Published in Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics, 2022
Nwadiuto (Diuto) Esiobu, Ifeoma M. Ezeonu, Francisca Nwaokorie
The shotgun sequencing technique is widely used in genome sequencing projects. Shotgun sequencing involves shearing large pieces of DNA into smaller fragments that are then sequenced randomly. These random fragments are then realigned and ordered into larger contiguous pieces that are representative of the original large DNA unit. The sequencing method is versatile and can be applied to study different domains of life – bacteria, fungi, protists and other eukaryotes in a sequencing reaction. This method will provide a more comprehensive view of all organisms represented within an ecosystem in one snap shop. It could be used to understand the functional analysis of a given niche when RNA-seq approach is sequenced.
Assays and enumeration of bioaerosols-traditional approaches to modern practices
Published in Aerosol Science and Technology, 2020
Maria D. King, Ronald E. Lacey, Hyoungmook Pak, Andrew Fearing, Gabriela Ramos, Tatiana Baig, Brooke Smith, Alexandra Koustova
More detailed identification of biodiversity and microbiome is possible with the advancement and combination of powerful tools. The most commonly used method to identify bacterial taxonomy is the 16S rRNA gene sequencing as this gene is present in most bacteria, its function has not changed over time, and it has a fairly long base pair of 1500 bp for analysis (Janda and Abbott 2007). This is advantageous in many ways, such as identifying the species of ambiguous isolates, but it also has its limitations. Some identified species strains are outdated and may not be accurate anymore, the sequencing resolution does not clearly distinguish between certain bacterial strains, and there are still some errors in public databases of sequences. Databases, including the open access GenBank (National Center for Biotechnology Information [NCBI]), the European Nucleotide Archive, the DNA Data Bank of Japan, and the UniProt, Protein Data Bank, Ensembl and InterPro are powerful tools that allow the search and alignment of nucleotide or protein sequences (Boratyn et al. 2013; Wheeler and Bhagwat 2007). Shotgun sequencing is another method of high-throughput sequencing that uses untargeted sequencing over a microbial genome to compile whole genomes. Although it has some limitations, such as high cost, lack of valid annotations, and genome biases, shotgun sequencing results in metagenomics that enables complex microorganisms to be identified and analyzed (Quince et al. 2017). Next-generation sequencing (NGS) platforms enable other types of microbiome analysis and identification, such as whole-genome sequencing, total RNA sequencing, and methylation sequencing (Illumina 2016). The NGS technologies enable the generation of millions of reads from aerosol samples to analyze the genomics and transcriptomics (for RNA analysis), and have been successfully used in several bioaerosol studies (Womack et al. 2015; Shin et al. 2015). Metagenomic approaches enable comprehensive determination of the diversity and metabolic potential of the collected airborne organisms.