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Molecular Diagnostic Solutions in Algal Cultivation Systems
Published in Stephen P. Slocombe, John R. Benemann, Microalgal Production, 2017
Laura T. Carney, Robert C. McBride, Val H. Smith, Todd W. Lane
Generally, sequencers fall into two broad categories, high-volume machines that are found in core facilities and commercial providers, and less expensive, lower volume “personal” sequencers. Personal sequencers—including the Illumina MiSeq, the 454 GS, and the Ion Torrent PGM—have the advantage of relatively rapid run times and lower cost per run than the larger machines. Pacific Biosciences sequencers are targeted for specific applications requiring a relatively small number of long reads. The costs associated with the actual sequencing can be reduced by the creation of multiplexed sequencing libraries allowing for the sequencing of more than one sample per lane. Various systems that take advantage of each type of sequencer have been developed for the creation of multiplexed libraries (McKenna et al. 2008; Caporaso et al. 2011; Whiteley et al. 2012).
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).
Current Use and Future Promise of Genetic Engineering
Published in Michael Hehenberger, Zhi Xia, Huanming Yang, Our Animal Connection, 2020
Michael Hehenberger, Zhi Xia, Huanming Yang
The ion torrent concept is based on a combination of DNA fragment amplification and electrical detection, replacing the optical detection of pyrophosphate used in pyrosequencing with electrical detection of the hydrogen ions (protons) which—as shown above in Fig. 7.2—are also released whenever a nucleotide is integrated.
Metagenome based analysis of groundwater from arsenic contaminated sites of West Bengal revealed community diversity and their metabolic potential
Published in Journal of Environmental Science and Health, Part A, 2023
Anumeha Saha, Abhishek Gupta, Pinaki Sar
Total DNA was extracted from 2 L of groundwater using Epicentre Metagenomic DNA isolation kit, following manufacturer protocol post filtration of groundwater through 0.1 µm membrane filter. DNA concentration was determined using NanoDrop 1000 spectrophotometer and Qubit 3.0 fluorometer (Invitrogen, Thermo Fisher Scientific). Post quantification, total DNA samples were subjected to amplification of the V4 hypervariable region of 16S rRNA gene using barcoded V4 specific primer set 515 F/806R.[33] PCR reaction was performed in 25 µL volume using Amplitaq Gold 360 Mastermix (Applied Biosystems), 40 pmol of barcoded forward and reverse primers each, 10-50 ng of metagenomic DNA under the following program: Initial denaturation 95 °C for 10 min followed by 35 cycles of 95 °C for 35 sec, 50 °C for 40 sec, 72 °C for 40 sec and a final extension at 72 °C for 10 min. PCR amplified products were run on 2% SizeSelect E-gel (Invitrogen). Amplicon library was prepared, further processed, and subjected to sequencing on in-house Ion Torrent Next-Generation (Ion S5) Sequencing platform using Ion 530™ Chip Kit and Ion 520™ and Ion 530™ Kit-OT2 (Thermo Fisher Scientific).
Use of plant materials for the bioremediation of soil from an industrial site
Published in Journal of Environmental Science and Health, Part A, 2020
Danielle Aparecida Duarte Nunes, Andrea Medeiros Salgado, Emanuela Forestieri da Gama-Rodrigues, Rodrigo Gouvêa Taketani, Cláudia Duarte da Cunha, Eliana Flávia Camporese Sérvulo
To evaluate the microbial diversity in the different bioremediation treatments tested, large-scale sequencing experiments of the gene encoding the ribosomal rRNA bacterial domain were performed. DNA was extracted using the commercial FastDNA extraction DNA spin kit for soil (MPBio), following the manufacturer’s instructions. After DNA extraction, the primers 967f and 1193r were used to amplify the genes encoding the bacteria 16S rRNA.[30,31] In the Ion Torrent sequencing PCR reactions, barcode sequences[32] were added to the 5’ end of the forward primers. In the same manner, specific adaptive sequences were added to the 5’ ends of the primers for the emulsion PCR amplification (emPCR), which is necessary for the Ion Torrent sequencing. Libraries for large-scale sequencing were prepared using the Ion PGM Template OT2 200pb kit and used according to the manufacturer’s protocol (Life Technologies). The sequencing was performed on the Ion torrent PGM system using the Ion PGM sequencing kit 200pb on the 316 v2 chip. The sequences obtained were analyzed using the Qiime platform[33] following the protocol described by Kavamura et al.34 All statistical analyses were also performed using this software through the core_diversity_analysis.py command.
Next-generation DNA sequencing of oral microbes at the Sir John Walsh Research Institute: technologies, tools and achievements
Published in Journal of the Royal Society of New Zealand, 2020
Nicholas C. K. Heng, Jo-Ann L. Stanton
In 2010, the first of the semiconductor sequencing systems, the Life Technologies Personal Genome Machine (PGM), was released (Rothberg et al. 2011). Instead of detecting photons or fluorescence, semiconductor (also known as ‘Ion Torrent’) sequencing involves detecting the subtle change in pH (translated to voltage) following the release of a proton (hydrogen ion) during the polymerisation of a DNA strand. Like 454- and Illumina–based sequencing, nucleotides are individually cycled during the sequencing run. Although homopolymers would theoretically result in proportional increases in pH/voltage, the Ion Torrent detection mechanism also suffered from the ‘homopolymer effect’, leading to sequencing errors. Nevertheless, the distinct advantage of Ion Torrent is speed as >1 Gbp of sequence data (200–400 bp read lengths) can be generated in a mere two hours.