Hert, D. G., Fredlake, C. P. & Barron, A. E. Advantages and limitations of next-generation sequencing technologies: a comparison of electrophoresis and non-electrophoresis methods. Electrophoresis 29, 4618-4626

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Electrophoresis (Impact Factor: 3.03). 12/2008; 29(23):4618-26. DOI: 10.1002/elps.200800456
Source: PubMed


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.

27 Reads
    • "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). "
    [Show abstract] [Hide abstract]
    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 · 1.59 Impact Factor
  • Source
    • "High-quality sequencing approaches have been suggested in conjunction with high-throughput sequencing for comparative genomics analyses and genome evolution studies (Alkan et al. [2011]). A technology which combines the massive throughput of the NGS with the long read lengths achieved by electrophoresis-based Sanger sequencing, would enable rapid, high-quality production of de novo genome sequences (Hert et al. [2008]). Future directions in the field of DNA sequencing are the ability to use individual molecules without any library preparation or amplification, the identification of specific nucleotide modifications, and generation of longer sequence reads (Kircher and Kelso [2010]). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Rice is a model system used for crop genomics studies. The completion of the rice genome draft sequences in 2002 not only accelerated functional genome studies, but also initiated a new era of resequencing rice genomes. Based on the reference genome in rice, next-generation sequencing (NGS) using the high-throughput sequencing system can efficiently accomplish whole genome resequencing of various genetic populations and diverse germplasm resources. Resequencing technology has been effectively utilized in evolutionary analysis, rice genomics and functional genomics studies. This technique is beneficial for both bridging the knowledge gap between genotype and phenotype and facilitating molecular breeding via gene design in rice. Here, we also discuss the limitation, application and future prospects of rice resequencing.
    Rice 07/2014; 7(1):4. DOI:10.1186/s12284-014-0004-7 · 3.92 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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 · 5.14 Impact Factor
Show more


27 Reads
Available from