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Understanding Microbial Communities
Published in Jean F. Challacombe, Metabolic Pathway Engineering, 2021
Third-generation sequencing technologies that produce long sequence reads are currently marketed by PacBio and Nanopore. The PacBio Sequel platforms (https://www.pacb.com) employ single molecule real-time (SMRT) technology involving a DNA polymerase that performs continuous template-directed synthesis using fluorescently labeled deoxyribonucleoside triphosphates [71]. The nanopore long read sequencing technology arose from research using nanopores to detect various molecules including DNA, RNA, and proteins [72, 73]. This technology was used by Oxford Nanopore Technologies to develop their single molecule real-time nanopore sequencers [74], which were introduced in 2012 [75]. Beginning with the two original nanopore platforms (GridlON and MinlON), the products offered by Oxford Nanopore Technologies (https://nanoporetech.com) have expanded to include the very small SmidglON, the larger scale PromethlON, and an adaptor called Flongle for MinlON or GridlON that enables sequencing on single-use flow cells.
Single-crystal silicon nanopore and arrays
Published in Klaus D. Sattler, Silicon Nanomaterials Sourcebook, 2017
To further reduce the cost, operation time, and equipment size of the DNA sequencing process, while continually increasing the contiguous read length, throughput, and accuracy, researchers have proposed different approaches for DNA sequencing [3]. This led to the emergence of the third-generation sequencing technologies, such as the real-time sequencing by synthesis technology and direct image technology. Nanopore-based DNA sequencing technology has become one of the most attractive and promising third-generation sequencing technologies because of its outstanding characteristics of label-free, amplification-free, great read length, and high throughput, which offer possibilities of high-quality gene sequencing applications, such as de novo sequencing, high-resolution analysis of chromosomal structure variation, and long-range haplotype mapping.
Advanced Molecular Tools and Techniques for Assessment of Microbial Diversity in Fermented Food Products
Published in Deepak Kumar Verma, Ami R. Patel, Sudhanshu Billoria, Geetanjali Kaushik, Maninder Kaur, Microbial Biotechnology in Food Processing and Health, 2023
Damanpreet Kaur, Sushma Gurumayum, Prasad Rasane, Sawinder Kaur, Jyoti Singh, Navneet Kaur, Kajal Dhawan, Ashwani Kumar
Although 454 pyrosequencing was the first commercially available system of NGS technology, but this system is now no longer in use (Claesson et al., 2010). However, Illumina and Ion Torrent are the latest next-generation technologies currently used. Both Illumina MiSeq and Ion Torrent PGM platforms are now increasingly being used to provide in-depth analysis of microbial diversity using 16S rRNA gene. Illumina sequencing amplifies the already prepared DNA fragments by isothermic bridge PCR and covalently links amplicons to solid surface and thus results in arrayed cluster formation. Sequencing of these clusters then take place through continuous cycles of single-base extension. This results in identification of incorporated base and the cleavage by terminators enable further cycles. In contrast, Ion Torrent sequencing first prepares DNA fragment using emulsion PCR and then amplifies the individual fragment and results in profiling of bacterial community. However, among both these platforms, ion torrent exhibits high sequencing error rates as compared to Illumina (Salipante et al., 2014). Beside these, PacBio sequencing is also a method of real-time sequencing and is commonly referred to as third-generation sequencing. PacBio sequencing offers the benefit of longer read lengths and long sequence runs. The long sequence reads help in identification of precise location and repetitive sequence regions within single reads. However, due to high error rates, high cost per base and low throughout, the application of this method is hindered. During this sequencing method, sequence information is captured during the replication process of the target DNA molecule (Rhoads and Au, 2015). The bacterial community of various fermented food products have been evaluated using different platforms of NGS technology. The microbial ecology of products like daqu, sourdough, pu-erh tea, Shanxi aged vinegar and Tibetian kefir has been exploited using platforms like Illumina MiSeq, Illumina HiSeq 2000 and pyrosequencing (He et al., 2017).
Metabolic modeling of synthetic microbial communities for bioremediation
Published in Critical Reviews in Environmental Science and Technology, 2023
Lvjing Wang, Xiaoyu Wang, Hao Wu, Haixia Wang, Yihan Wang, Zhenmei Lu
The combination of meta-omics and GEMs can predict the relationships among microorganisms in complex microbial communities. The reconstruction of high-quality GEMs for FBA begins with metagenomic well-assembled genomes. Nevertheless, random sequencing enables de novo identification of uncultured microorganisms, trustworthy annotation, and gene identification. Additionally, more recent and promising techniques, such as "third-generation sequencing," have been shown to enhance the assembly quality and completeness values of recovered genomes, leading to an in-depth understanding of a variety of microbiomes (De Bernardini et al., 2022). Integrating metagenomic binning with GEMs provided a deeper understanding of the potential syntrophic interaction among five dominant species within biofilm samples from an anaerobic biogas reactor (De Bernardini et al., 2022). Moreover, researchers proposed a meta-network approach in which the levels of protein expression and taxonomic designations of proteins are used as the most pertinent cues for inferring an active set of reactions (Tobalina et al., 2015). This approach was applied to draft context-specific GEMs for 2 different naphthalene-degrading communities with or without bio-simulation. It was found that the experimental results agreed well with the computational predictions. In summary, the development of omics data integration methods for GEMs led to an expansion of the application domain for GEMs by allowing GEMs to be customized in accordance with specific contexts.
Culturing the uncultured microbial majority in activated sludge: A critical review
Published in Critical Reviews in Environmental Science and Technology, 2023
In recent years, long reads sequencing (or third-generation sequencing) has received increased attention with two currently dominant technologies: single-molecule real-time sequencing from PacBio and Nanopore sequencing. Compared with short reads sequencing, long reads sequencing could provide highly resolved phylogenetic classification and detect base modifications thus getting increased appealing in microbiology (Lam et al., 2020; Leggett et al., 2020; Matsuo et al., 2021). While the widespread application of long reads sequencing is impeded by the high error rate (∼1% for Pacbio (Wenger et al., 2019) and ∼5% for Nanopore (Jain et al., 2018)). To overcome this issue, manufacturers update the chemicals to improve accuracy, and researchers design appropriate correction tools (like Racon (Vaser et al., 2017) and Pilon (Walker et al., 2014)) or combine molecular methods like unique molecular identifiers (UMIs) (Islam et al., 2014; Kivioja et al., 2012) to get final consensus sequences. For example, the NanoCLUST pipeline was developed for the correction and classification of full-length 16S rRNA nanopore reads (Rodríguez-Pérez et al., 2021), and researchers introduced UMIs into Pacbio and Nanopore sequencing to generate whole rRNA operon with a mean error rate <0.01% (Karst et al., 2021).