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1.
Methods Mol Biol ; 1347: 15-41, 2015.
Article in English | MEDLINE | ID: mdl-26374307

ABSTRACT

Whole genome amplification (WGA) is a widely used molecular technique that is becoming increasingly necessary in genetic research on a range of sample types including individual cells, fossilized remains and entire ecosystems. Multiple methods of WGA have been developed, each with specific strengths and weaknesses, but with a common defect in that each method distorts the initial template DNA during the course of amplification. The type, extent, and circumstance of the bias vary with the WGA method and particulars of the template DNA. In this review, we endeavor to discuss the types of bias introduced, the susceptibility of common WGA techniques to these bias types, and the interdependence between bias and characteristics of the template DNA. Finally, we attempt to illustrate some of the criteria specific to the analytical platform and research application that should be considered to enable combination of the appropriate WGA method, template DNA, sequencing platform, and intended use for optimal results.


Subject(s)
Genome , Genomics/methods , Genomics/standards , Nucleic Acid Amplification Techniques/standards , Animals , Artifacts , Bias , Humans
2.
J Mol Diagn ; 15(2): 171-6, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23274167

ABSTRACT

There is growing demand for routine identification of actionable mutations in clinical cancer specimens. Genotyping platforms must provide rapid turnaround times and work effectively with limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissue specimens that often yield poor quality DNA. We describe semiconductor-based sequencing of DNA from FFPE specimens using a single-tube, multiplexed panel of 190 amplicons targeting 46 cancer genes. With just 10 ng of input DNA, average read depths of 2000× can be obtained in 48 hours, with >95% of the reads on target. A validation set of 45 FFPE tumor specimens containing 53 point mutations previously identified with a mass spectrometry-based genotyping platform, along with 19 indels ranging from 4 to 63 bp, was used to evaluate assay performance. With a mutant allele ratio cutoff of 8%, we were able to achieve 100% sensitivity (95% CI = 97.3% to 100.0%) and 95.1% specificity (95% CI = 91.8% to 98.0%) of point mutation detection. All indels were visible by manual inspection of aligned reads; 6/9 indels ≤12 bp long were detected by the variant caller software either exactly or as mismatched nucleotides within the indel region. The rapid turnaround time and low input DNA requirements make the multiplex PCR and semiconductor-based sequencing approach a viable option for mutation detection in a clinical laboratory.


Subject(s)
Genotyping Techniques , Multiplex Polymerase Chain Reaction , Neoplasms/genetics , Semiconductors , Sequence Analysis, DNA , Gene Library , Genetic Variation , Genotype , Humans , Mutation , Neoplasms/diagnosis , ROC Curve
3.
Nature ; 475(7356): 348-52, 2011 Jul 20.
Article in English | MEDLINE | ID: mdl-21776081

ABSTRACT

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.


Subject(s)
Genome, Bacterial/genetics , Genome, Human/genetics , Genomics/instrumentation , Genomics/methods , Semiconductors , Sequence Analysis, DNA/instrumentation , Sequence Analysis, DNA/methods , Escherichia coli/genetics , Humans , Light , Male , Rhodopseudomonas/genetics , Vibrio/genetics
4.
Nat Biotechnol ; 26(10): 1117-24, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18846085

ABSTRACT

The 454 Sequencer has dramatically increased the volume of sequencing conducted by the scientific community and expanded the range of problems that can be addressed by the direct readouts of DNA sequence. Key breakthroughs in the development of the 454 sequencing platform included higher throughput, simplified all in vitro sample preparation and the miniaturization of sequencing chemistries, enabling massively parallel sequencing reactions to be carried out at a scale and cost not previously possible. Together with other recently released next-generation technologies, the 454 platform has started to democratize sequencing, providing individual laboratories with access to capacities that rival those previously found only at a handful of large sequencing centers. Over the past 18 months, 454 sequencing has led to a better understanding of the structure of the human genome, allowed the first non-Sanger sequence of an individual human and opened up new approaches to identify small RNAs. To make next-generation technologies more widely accessible, they must become easier to use and less costly. In the longer term, the principles established by 454 sequencing might reduce cost further, potentially enabling personalized genomics.


Subject(s)
Chromosome Mapping/instrumentation , Genome, Human/genetics , Genomics/instrumentation , Sequence Analysis, DNA/instrumentation , Equipment Design , Equipment Failure Analysis , Humans , Sequence Analysis, DNA/methods , Technology Assessment, Biomedical
5.
Anal Chem ; 80(23): 8975-81, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-19551929

ABSTRACT

Limiting dilution PCR has become an increasingly useful technique for the detection and quantification of rare species in a population, but the limit of detection and accuracy of quantification are largely determined by the number of reactions that can be analyzed. Increased throughput may be achieved by reducing the reaction volume and increasing processivity. We have designed a high-throughput microfluidic chip that encapsulates PCR reagents in millions of picoliter droplets in a continuous oil flow. The oil stream conducts the droplets through alternating denaturation and annealing zones, resulting in rapid (55-s cycles) and efficient PCR amplification. Inclusion of fluorescent probes in the PCR reaction mix permits the amplification process to be monitored within individual droplets at specific locations within the microfluidic chip. We show that amplification of a 245-bp adenovirus product can be detected and quantified in 35 min at starting template concentrations as low as 1 template molecule/167 droplets (0.003 pg/microL). The frequencies of positive reactions over a range of template concentrations agree closely with the frequencies predicted by Poisson statistics, demonstrating both the accuracy and sensitivity of this platform for limiting dilution and digital PCR applications.


Subject(s)
Adenoviridae/genetics , DNA, Viral/analysis , Microfluidic Analytical Techniques/instrumentation , Polymerase Chain Reaction/methods , Base Sequence , DNA Primers/genetics , Equipment Design , Fluorescent Dyes , Genome, Viral , Microfluidic Analytical Techniques/economics , Microfluidic Analytical Techniques/methods , Polymerase Chain Reaction/economics , Polymerase Chain Reaction/instrumentation , Sample Size , Sensitivity and Specificity
7.
BMC Genomics ; 7: 216, 2006 Aug 23.
Article in English | MEDLINE | ID: mdl-16928277

ABSTRACT

BACKGROUND: Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs) or large scale (CGH array, FISH) methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls. RESULTS: All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR. CONCLUSION: Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.


Subject(s)
Bias , Genomics/methods , Nucleic Acid Amplification Techniques , Sequence Analysis, DNA/methods , Campylobacter jejuni/genetics , Chromosomes, Bacterial , DNA Probes , Genome, Bacterial , Genomics/statistics & numerical data , Halobacterium/genetics , Statistics, Nonparametric
8.
Nat Methods ; 3(7): 541-3, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16791212

ABSTRACT

Three protocols in this issue highlight applications of emulsification procedures, which deliver high-throughput potential to the molecular biology laboratory, without the need for automation. These procedures have already generated interesting results and spurred the development of exciting new technologies, while requiring only readily available laboratory equipment.


Subject(s)
Biology/methods , Bioreactors , Emulsions , Microchip Analytical Procedures , Research Design
9.
Nature ; 437(7057): 376-80, 2005 Sep 15.
Article in English | MEDLINE | ID: mdl-16056220

ABSTRACT

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.


Subject(s)
Genome, Bacterial , Genomics/instrumentation , Microchemistry/instrumentation , Mycoplasma genitalium/genetics , Sequence Analysis, DNA/instrumentation , Electrophoresis, Capillary , Emulsions , Fiber Optic Technology , Genomics/economics , Microchemistry/economics , Polymerase Chain Reaction , Reproducibility of Results , Sensitivity and Specificity , Sequence Analysis, DNA/economics , Time Factors
10.
Electrophoresis ; 24(21): 3769-77, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14613204

ABSTRACT

We demonstrate successful, simultaneous polymerase chain reaction (PCR) amplification of up to 300 000 discrete reactions in a novel platform, the PicoTiterPlate. In addition to elevated throughput, the PicoTiterPlate based amplifications (PTPCR) can be performed in extremely small volumes: individual reactions volumes are as low as 39.5 pL, with a total 15.3 microL reaction volume for the entire PicoTiterPlate. The bulk PTPCR product can be recovered and assayed with real-time PCR, or discrete PTPCR products can be driven to solid supports, enabling downstream applications such as translation/transcription or sequencing.


Subject(s)
DNA/chemistry , Miniaturization/instrumentation , Polymerase Chain Reaction/methods , Base Sequence , DNA/genetics , DNA Primers , Fiber Optic Technology , Microscopy, Electron, Scanning , Miniaturization/methods , Nucleic Acid Hybridization/methods , Sensitivity and Specificity
11.
Genome Res ; 13(2): 294-307, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12566408

ABSTRACT

Structural genetic alterations in cancer often involve gene loss or gene amplification. With the advent of microarray approaches for the analysis of the genome, as exemplified by array-CGH (Comparative Genomic Hybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density. However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array-CGH analysis. We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples. We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias. The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours. Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines. Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples. Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material.


Subject(s)
DNA/analysis , Genome , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line , Chromosomes, Artificial, Bacterial/genetics , DNA/genetics , DNA, Complementary/genetics , DNA, Fungal/analysis , DNA, Fungal/genetics , Gene Expression Profiling/methods , Genetic Markers/genetics , Genome, Fungal , Humans , Lymphocytes/chemistry , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Hybridization/methods , Oligonucleotide Array Sequence Analysis/methods , Polyploidy , Saccharomyces cerevisiae/genetics , Tumor Cells, Cultured
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