Article

Sequencing Technologies – The Next Generation

Human Genome Sequencing Center and Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
Nature Reviews Genetics (Impact Factor: 39.79). 12/2009; 11(1):31-46. DOI: 10.1038/nrg2626
Source: PubMed

ABSTRACT Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.

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Available from: Supriya Karkra, Aug 24, 2015
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    • "Key to the success of such endeavors is having the correct number of genetic markers to accurately associate phenotypes with genotypes, or to characterize population parameters (e.g., inbreeding). With the advent of highthroughput sequencing technology (Metzker 2010), continuing decline in sequencing costs (Glenn 2011), and new methods for simultaneously generating novel loci and genotypes (Baird et al. 2008; Hohenlohe et al. 2010; Elshire et al. 2011), large panels of markers can now be developed for most species. However, one key parameter that has received relatively little attention in the conservation community is linkage disequilibrium (LD). "
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    ABSTRACT: Linkage disequilibrium (LD) is the nonrandom association of alleles at two markers. Patterns of LD have biological implications as well as practical ones when designing association studies or conservation programs aimed at identifying the genetic basis of fitness differences within and among populations. However, the temporal dynamics of LD in wild populations has received little empirical attention. In this study, we examined the overall extent of LD, the effect of sample size on the accuracy and precision of LD estimates, and the temporal dynamics of LD in two populations of bighorn sheep (Ovis canadensis) with different demographic histories. Using over 200 microsatellite loci, we assessed two metrics of multi-allelic LD, D′, and χ′2. We found that both populations exhibited high levels of LD, although the extent was much shorter in a native population than one that was founded via translocation, experienced a prolonged bottleneck post founding, followed by recent admixture. In addition, we observed significant variation in LD in relation to the sample size used, with small sample sizes leading to depressed estimates of the extent of LD but inflated estimates of background levels of LD. In contrast, there was not much variation in LD among yearly cross-sections within either population once sample size was accounted for. Lack of pronounced interannual variability suggests that researchers may not have to worry about interannual variation when estimating LD in a population and can instead focus on obtaining the largest sample size possible.
    Ecology and Evolution 08/2015; 5(16):3401-3412. DOI:10.1002/ece3.1612 · 1.66 Impact Factor
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    • "The Illumina ⁄ Solexa digital gene expression (DGE) system is an improved tag-based method that can sequence, in parallel, millions of DNA molecules that are derived directly from mRNA [15]. The DGE system enables the sequencing of total cDNA for the derivation of an accurate estimate of gene expression, both individually and comprehensively, and the discovery of novel regions of transcription. "
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    ABSTRACT: were grouped into 43 functional categories involved in seven significant pathways. The most enriched categories were those that were populated by transcripts involved in metabolism, signal transduction and cellular transport. Many genes and/or biological pathways were found to be common among the three libraries, for example, genes participating in transport, stress response, auxin transport and signaling, etc. Next, the expression patterns of 12 genes were assessed with quantitative real-time PCR, the results of which agreed with the Solexa analysis. In conclusion, we revealed complex changes in the transcriptome during tobacco root development related to drought resistance, and provided a comprehensive set of data that is essential to understanding the molecular regulatory mechanisms involved. These data may prove valuable in future studies of the molecular mechanisms regulating root development in response to drought stress in tobacco and other plants.
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    • "Traditional Sanger sequencing analysis on tissue biopsy has been widely used to guide therapy for cancer patients. Nevertheless, next-generation sequencing (NGS) technology holds a number of advantages over traditional methods, including the ability to fully interrogate large numbers of samples/genes/mutations in a single run, higher throughput, sensitivity and specificity, and automation-friendly [7] [8]. The rapid advances in NGS technology will further lower the overall cost, speed the turnaround time, increase the breadth and accuracy of genome sequencing, detect important genomic parameters, and most importantly, become applicable to lower-quantity and poor-quality specimens [9]. "
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    ABSTRACT: The non-invasive circulating cell-free DNA (cfDNA) approach – a liquid biopsy – is revolutionizing a paradigm shift in how cancer is detected, monitored and treated. In contrast to single-site, single time-point sampling by tissue biopsy, real-time and longitudinal mutation profile derived from tumor-specific cfDNA could potentially inform better and faster clinical decision-making, monitor tumor dynamics, assess response to treatment and identify mutations associated with acquired drug resistance. However, cfDNA analysis requires large volume of blood due to its relatively low amount in circulation and poor extraction efficiency of current methodologies. To overcome these major challenges, we have developed a proprietary cfDNA recovery technology with unique features of ultra-low input and ultra-high output. In this study, we evaluated our method side-by-side with the industry standard Qiagen kit, for the yield, cfDNA amplifiability and mutation detection from patient plasma. Compared to Qiagen cfDNA extraction kit using different chemistry and different workflow, our approach allowed high-yield cfDNA enrichment directly from droplet volumes of unprocessed plasma, leading to >100-fold more recovery. NGS studies with cfDNA from 17 cancer plasma and 2 spiked samples further demonstrated the superiority of our protocol over Qiagen kit in generating more usable, on-target, high-quality ≥Q20 reads, and detecting more mutations. Our cfDNA preparation breakthrough enables clinicians and laboratories to work with a sample volume as small as 20 microliters (via a finger-prick), in contrast to the current requirement of 10-20 milliliters, further expedite clinical decision-making and identify targeted therapies for eligible patients in a time-and cost-efficient manner.
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