Advantages and limitations of next-generation sequencing technologies: A comparison of electrophoresis and non-electrophoresis methods

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Electrophoresis (Impact Factor: 3.16). 12/2008; 29(23):4618-26. DOI: 10.1002/elps.200800456
Source: PubMed

ABSTRACT The reference human genome provides an adequate basis for biological researchers to study the relationship between genotype and the associated phenotypes, but a large push is underway to sequence many more genomes to determine the role of various specificities among different individuals that control these relationships and to enable the use of human genome data for personalized and preventative healthcare. The current electrophoretic methodology for sequencing an entire mammalian genome, which includes standard molecular biology techniques for genomic sample preparation and the separation of DNA fragments using capillary array electrophoresis, remains far too expensive ($5 million) to make genome sequencing ubiquitous. The National Human Genome Research Institute has put forth goals to reduce the cost of human genome sequencing to $100,000 in the short term and $1000 in the long term to spur the innovative development of technologies that will permit the routine sequencing of human genomes for use as a diagnostic tool for disease. Since the announcement of these goals, several companies have developed and released new, non-electrophoresis-based sequencing instruments that enable massive throughput in the gathering of genomic information. In this review, we discuss the advantages and limitations of these new, massively parallel sequencers and compare them with the currently developing next generation of electrophoresis-based genetic analysis platforms, specifically microchip electrophoresis devices, in the context of three distinct types of genetic analysis.

    • "Since the advent of environmental DNA-based techniques , these contemporary methods have significantly gained impetus over traditional approaches, mainly because the latter have reportedly underestimated diversity. The advantages and disadvantages of the former techniques have been well discussed (Hert et al. 2008; Git et al. 2010). "
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    ABSTRACT: The molecular diversity of freshwater microeukaryotes, particularly phytoplankton, in the Arctic Svalbard, has been relatively unexplored. Freshwater algae are considered biological indicators of environmental change and can be useful in assessing the impact of global climate change and increased environmental pollution. In this study, freshwater microeukaryotes in an Arctic reservoir at Ny-Ǻlesund (Svalbard, Norway) were studied using the hypervariable V1–V3 small subunit rRNA and 454 pyrosequencing. On the basis of 8,956 reads, we revealed high genetic diversity in eukaryotes, representing all known eukaryotic supergroups, except Excavata. “Chromalveolata” (previously supergroup Chromalveolata) and Archaeplastida were the most and least abundant supergroups, respectively. After data mining, 57 phylotypes were detected from 7,398 pyrosequences. They were dominated by stramenopiles (84 %) and Dinoflagellata (13 %), with minor contributions from Cryptophyta, Chlorophyta, and Telonemida. The detection of algae belonging to the orders Mamiellales and Monomastigales provides a window into a fraction of the ‘rare biosphere’ that had previously been undetected in such environments. Interestingly, no haptophytes were recorded. Stramenopiles and Dinoflagellata mainly comprised taxa belonging to the families Chrysophyceae, Synurophyceae, and Dinophyceae. On the basis of the proportion of operational taxonomic units, the dominant phylotypes were found to include Ochromonas spp., Mallomonas spp., and Uroglena americana. These results demonstrate the significance of a chrysophyte-dominated microeukaryotic community, which is of great potential for future studies in terms of reconstruction of past climate trends and monitoring of environmental change in the Arctic.
    Polar Biology 02/2015; 38(2):179-87. DOI:10.1007/s00300-014-1576-9 · 2.07 Impact Factor
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    • "Although this is one particular study and the error rate may dramatically reduce with the rigorous application of quality controls and standards to be defined, next-generation sequencing may remain of limited use for immediate forensic applications. Cautions are mainly about: the quantity and quality of DNA required; the persistence of PCR bias as forensic applications is expected to use target sequencing approaches (not whole genome); the risk of cross contamination is due to a large number of parallel reactions; the difficulty of repeat sequence analysis (STR) and finally; the times, costs, and expertise required for this type of analysis [Berglund et al., 2011; Hert et al., 2008; Metzker, 2010; Snyder et al., 2011]. With these considerations in mind, we propose here new genetic markers, the DIP–STR, which are located throughout the genome, highly polymorphic, easy to genotype, and capable of resolving extremely unbalanced two DNA mixtures (ratio 1:1,000). "
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    ABSTRACT: Samples containing highly unbalanced DNA mixtures from two individuals commonly occur in forensic mixed stains and in peripheral blood DNA microchimerism during pregnancy or following organ transplant. Because of PCR amplification bias, the genetic identification of a DNA that contributes trace amounts to a mixed sample represents a tremendous challenge. This means that standard genetic markers, namely microsatellites, also referred to as Short Tandem Repeats (STR), and Single Nucleotide Polymorphism (SNP) have limited power in addressing common questions of forensic and medical genetics. To address this issue, we developed a molecular marker, named DIP-STR that relies on pairing deletion/insertion polymorphisms (DIP) with STR. This novel analytical approach allows for the unambiguous genotyping of a minor component in the presence of a major component, where DIP-STR genotypes of the minor were successfully procured at ratios up to 1:1000. The compound nature of this marker generates a high level of polymorphism that is suitable for identity testing. Here, we demonstrate the power of the DIP-STR approach on an initial set of 9 markers surveyed in a Swiss population. Finally, we discuss the limitations and potential applications of our new system including preliminary tests on clinical samples and estimates of their performance on simulated DNA mixtures.
    Human Mutation 02/2013; 34(4). DOI:10.1002/humu.22280 · 5.05 Impact Factor
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    • "This first-generation technology is based on incorporation of fluorescently labeled deoxynucleoside triphosphate (dNTP) and primers into a PCR that set the stage for automated high-throughput DNA sequencing. With the information obtained from the last terminator base in the 4 individual base reaction tubes after size separation , the original sequence could be determined (Venter et al., 2001; Hert et al., 2008; Schloss, 2008). Around at the same time in 1977, Maxam and Gilbert developed a chemical degradation DNA sequencing method in which terminally labeled DNA fragments were chemically cleaved at specific bases and separated by gel electrophoresis (Maxam and Gilbert, 1977). "
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    ABSTRACT: The era of molecular biology and automation of the Sanger chain-terminator sequencing method has led to discovery and advances in diagnostics and biotechnology. The Sanger methodology dominated research for over 2 decades, leading to significant accomplishments and technological improvements in DNA sequencing. Next-generation high-throughput sequencing (HT-NGS) technologies were developed subsequently to overcome the limitations of this first generation technology that include higher speed, less labor, and lowered cost. Various platforms developed include sequencing-by-synthesis 454 Life Sciences, Illumina (Solexa) sequencing, SOLiD sequencing (among others), and the Ion Torrent semiconductor sequencing technologies that use different detection principles. As technology advances, progress made toward third generation sequencing technologies are being reported, which include Nanopore Sequencing and real-time monitoring of PCR activity through fluorescent resonant energy transfer. The advantages of these technologies include scalability, simplicity, with increasing DNA polymerase performance and yields, being less error prone, and even more economically feasible with the eventual goal of obtaining real-time results. These technologies can be directly applied to improve poultry production and enhance food safety. For example, sequence-based (determination of the gut microbial community, genes for metabolic pathways, or presence of plasmids) and function-based (screening for function such as antibiotic resistance, or vitamin production) metagenomic analysis can be carried out. Gut microbialflora/communities of poultry can be sequenced to determine the changes that affect health and disease along with efficacy of methods to control pathogenic growth. Thus, the purpose of this review is to provide an overview of the principles of these current technologies and their potential application to improve poultry production and food safety as well as public health.
    Poultry Science 02/2013; 92(2):562-72. DOI:10.3382/ps.2012-02741 · 1.67 Impact Factor
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