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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    • "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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    • "NGS technologies have enabled whole-genome sequencing to identify potential phenotype-altering mutations. However, despite the decreased cost of sequencing, whole-genome sequencing remains costly [Metzker, 2010; Sulonen et al., 2011]. Further, for cancer research applications, higher depth of coverage is typically desired due to tumor samples having admixtures with normal tissue and tumor heterogeneity. "
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    ABSTRACT: Next generation sequencing (NGS) has aided characterization of genomic variation. While whole genome sequencing may capture all possible mutations, whole exome sequencing remains cost-effective and captures most phenotype-altering mutations. Initial strategies for exome enrichment utilized a hybridization-based capture approach. Recently, amplicon-based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture-based and two amplicon-based whole exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on-target alignment, uniformity, and variant calling. While the amplicon methods had higher on-target rates, the hybridization capture-based approaches demonstrated better uniformity. All methods identified many of the same single nucleotide variants, but each amplicon-based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform-specific variant calling algorithms. All methods demonstrated effective copy number variant calling when evaluated against a single nucleotide polymorphism array. This study illustrates some differences between whole exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Human Mutation 06/2015; DOI:10.1002/humu.22825
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    • "under an umbrella term of next - generation sequencing ( NGS ) . ( Mardis , 2013 ; Metzker , 2010 "
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    ABSTRACT: The past couple of decades have witnessed global resurgence of herbal-based health care. As a result, the trade of raw drugs has surged globally. Accurate and fast scientific identification of the plant(s) is the key to success for the herbal drug industry. The conventional approach is to engage an expert taxonomist, who uses a mix of traditional and modern techniques for precise plant identification. However, for bulk identification at industrial scale, the process is protracted and time-consuming. DNA barcoding, on the other hand, offers an alternative and feasible taxonomic tool box for rapid and robust species identification. For the success of DNA barcode, the barcode loci must have sufficient information to differentiate unambiguously between closely related plant species and discover new cryptic species. For herbal plant identification, matK, rbcL, trnH-psbA, ITS, trnL-F, 5S-rRNA and 18S-rRNA have been used as successful DNA barcodes. Emerging advances in DNA barcoding coupled with next-generation sequencing and high-resolution melting curve analysis have paved the way for successful species-level resolution recovered from finished herbal products. Further, development of multilocus strategy and its application has provided new vistas to the DNA barcode-based plant identification for herbal drug industry. For successful and acceptable identification of herbal ingredients and a holistic quality control of the drug, DNA barcoding needs to work harmoniously with other components of the systems biology approach. We suggest that for effectively resolving authentication challenges associated with the herbal market, DNA barcoding must be used in conjunction with metabolomics along with need-based transcriptomics and proteomics. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
    Plant Biotechnology Journal 06/2015; DOI:10.1111/pbi.12419
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