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Analysis of Emerging Microbial Contaminants through Next-Generation Sequencing (NGS)
Published in Vineet Kumar, Vinod Kumar Garg, Sunil Kumar, Jayanta Kumar Biswas, Omics for Environmental Engineering and Microbiology Systems, 2023
Ayushman Gadnayak, Swayamprabha Sahoo, Ananya Nayak, Maheswata Sahoo, Sushma Dave, Jayashankar Das
Next-generation sequencing (NGS) technology is associated with an advanced technology that has been responsible for sequencing the whole genome of an organism. This technology is responsible for formulating criteria for analyzing the genetic variation present within a DNA or RNA sequence. The process of NGS technology in sequencing thousands of smaller fragments of genes in a sample is related to the analysis of the contaminated particles. Microbial contamination is one of the potential elements found through the processing of NGS technology to sequence the genetic material of a particular sample. The high-throughput NGS technology can efficiently analyze the microscopic contaminants of microorganisms from the sample. The study sheds light on the prospects of NGS on analyzing the emerging microbial contaminants through addressing the advantages.
Molecular Biology and Bioinformatics in Industrial Microbiology and Biotechnology
Published in Nduka Okafor, Benedict C. Okeke, Modern Industrial Microbiology and Biotechnology, 2017
Nduka Okafor, Benedict C. Okeke
For Sanger sequencing, a single strand of the DNA to be sequenced is mixed with a primer, DNA polymerase I, an excess of normal nucleotide triphosphates and a limiting (about 5%) of the ddNTP labeled with a fluorescent dye. Each ddNTP is labeled with a different fluorescent dye color. This primer will determine the starting point of the sequence being read and the direction of the sequencing reaction. DNA synthesis begins with the primer and terminates in a DNA chain when ddNTP is incorporated in place of normal dNTP. As all four normal nucleotides are present, chain elongation proceeds normally, until by chance, DNA polymerase inserts a dideoxy nucleotide instead of the normal deoxy nucleotide. The result is a series of fragments of varying lengths. Each of the four nucleotides is run separately with the appropriate ddNTP. The mix with the ddCTP produces fragments with C (cytosine) terminal; that with ddTTP (thymine) produces fragments with T terminals, etc. The fluorescent strands are separated from the DNA template and electrophoresed on a polyacrylamide gel to separate them according their lengths. If the gel is read manually, four lanes are prepared, one for each of the four reaction mixes. In an automated system, all four are mixed in one reaction and electrophoresed together. As the ddNTPs are of different colors, a scanner can scan the gel and record each color (nucleotide) separately. The Sanger method is used for relatively short fragments of DNA, about 700–800 nucleotides.
Foundation of Big Data and Internet of Things
Published in Vijayalakshmi Saravanan, Alagan Anpalagan, T. Poongodi, Firoz Khan, Securing IoT and Big Data, 2020
Vijayalakshmi Saravanan, Mark Nuneviller, Anju S. Pillai, Alagan Anpalagan
The first human genome was sequenced in 2003 at a cost of $2.7 billion, called the Human Genome Project. It aimed to discover the base pairs that make up human DNA and was one of the novel scientific research findings. This massive achievement opened the door to greater understanding of human genes that could lead to tremendous advancements in the treatment of diseases. In future, it is possible that medicine will be “personalized” to a patient’s individual genetic profile. In 2006, just three years after the first human genome was sequenced, Illumina sequenced a genome on its first machine for $300,000. Today, the cost has fallen to $1000, with the expectation that sequencing could be done for $100 in just a few years.
Culturing the uncultured microbial majority in activated sludge: A critical review
Published in Critical Reviews in Environmental Science and Technology, 2023
Sanger sequencing developed in 1977 is a method to sequence DNA using electrophoresis based on DNA polymerase's random incorporation of chain-terminating dideoxynucleotides during in vitro DNA replication. Sanger sequencing is still a widely used method now although it has been largely replaced by high-throughput sequencing methods in recent years, particularly for large-scale automated genome analyses. In cultivation studies, Sanger method is used in the final identification of a single colony or isolate as it can produce DNA sequence reads of >500 nucleotides and maintains a very low error rate (>99.99%). The new species is identified if its similarity of 16S rRNA gene with cultured species is below 98.65 (Kim et al., 2014; Yarza et al., 2014), and the current commonly used 16S rRNA databases include NCBI (Sayers et al., 2022), Silva (Quast et al., 2013), Greengene (DeSantis et al., 2006), and EzBioCloud (Yoon et al., 2017). Once the new species are confirmed, the corresponding steps should be followed for new species announcement, such as the brief description of sources and growth conditions of isolates, a panel of experiments to evaluate the morphological and biochemical characteristics, genotypic and phenotypic information for taxonomy classification, and constructions of GenBank 16S rRNA accession numbers and an assembly of strain numbers to promote the propagation of novel species.
Advanced 5D logistic and DNA encoding for medical images
Published in The Imaging Science Journal, 2023
Bharti Ahuja, Rajesh Doriya, Sharad Salunke, Md. Farukh Hashmi, Aditya Gupta
The technique of mapping the nucleotide sequence comprising a DNA strand is termed ‘DNA sequencing’. The four bases that make up genetic coding are: adenine (A), thymine (T), guanine (G), and cytosine (C). ‘A’ binds with ‘T’ and ‘G’ binds with ‘C’ [39]. We understand that each pixel in a digital image can also be represented using 8-bit binary integers [40]. The complementary digital numbers of the pair 0–3 and 1–2 are used for the four deoxynucleotides ‘A,’ ‘T,’ ‘G,’ and ‘C’ and their combinations to express the digital numbers ‘00,’ ‘11,’ ‘01,’ and ‘10’. For instance, any pixel value of 206 can be expressed in the corresponding binary as ‘11001110’. This binary is ‘TATC’ in DNA coding with the above rules. The four nucleotides can be combined in 24 different ways. Only eight encoding permutations are, though, compatible with the complementary principle. Figure 5, illustrates and Table 1 summarizes these rules.
Genomics is here: what can we do with it, and what ethical issues has it brought along for the ride?
Published in The New Bioethics, 2023
Fast forward to the present, however, and we live in a very different world. DNA sequencing is no longer done using the time-consuming and somewhat cumbersome Sanger method, which had been the cornerstone of the HGP. A series of ingenious technological advances, sometimes termed ‘next-generation sequencing’, combined with developments in computing, have dramatically cut both the time needed to compile the entire genome of a specific individual, but also the associated cost. By 2010, it was estimated that DNA could already be sequenced 50,000 times faster than it had a decade earlier (Venter 2010) and in 2022 it was reported that a team at Stanford University achieved the complete sequencing of a human genome in five hours and two minutes, facilitating the diagnosis of a rare cancer in under eight hours from initial receipt of the blood sample (Armitage 2022). Likewise, the costs of sequencing a complete human genome have fallen from some 10 million US Dollars in 2007 to less than $1000 per genome by 2021 (NIH 2021).