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

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.

[1]  D. Ehrlich,et al.  Optimization of high-performance DNA sequencing on short microfabricated electrophoretic devices. , 2000, Analytical chemistry.

[2]  Y. Rogers,et al.  Genomics: Massively parallel sequencing , 2005, Nature.

[3]  D. Wildman,et al.  Distinct genomic signatures of adaptation in pre- and postnatal environments during human evolution , 2008, Proceedings of the National Academy of Sciences.

[4]  Richard A Mathies,et al.  Microchip bioprocessor for integrated nanovolume sample purification and DNA sequencing. , 2002, Analytical chemistry.

[5]  Timothy B. Stockwell,et al.  The Sequence of the Human Genome , 2001, Science.

[6]  Evan Powell,et al.  Comparative Genomic Analyses of Seventeen Streptococcus pneumoniae Strains: Insights into the Pneumococcal Supragenome , 2007, Journal of bacteriology.

[7]  Gabor T. Marth,et al.  Whole-genome sequencing and variant discovery in C. elegans , 2008, Nature Methods.

[8]  James P. Landers,et al.  Developments toward a complete micro-total analysis system for Duchenne muscular dystrophy diagnosis , 2003 .

[9]  Z. Selvanayagam,et al.  Application of expression genomics in drug development and genomic medicine , 2004 .

[10]  S. Turner,et al.  Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations , 2003, Science.

[11]  P. McEwan,et al.  Eight hundred-base sequencing in a microfabricated electrophoretic device. , 2000, Analytical chemistry.

[12]  S. Jacobson,et al.  Multiple sample PCR amplification and electrophoretic analysis on a microchip. , 1998, Analytical chemistry.

[13]  Mark Gerstein,et al.  New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. , 2007, Genes & development.

[14]  Richard A Mathies,et al.  High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. V. Moran,et al.  Initial sequencing and analysis of the human genome. , 2001, Nature.

[16]  Haixu Tang,et al.  Fragment assembly with short reads , 2004, Bioinform..

[17]  E. Lander,et al.  Genomic mapping by fingerprinting random clones: a mathematical analysis. , 1988, Genomics.

[18]  Graham Bell,et al.  Phenotypic consequences of 1,000 generations of selection at elevated CO2 in a green alga , 2004, Nature.

[19]  R. Mathies,et al.  Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis. , 2001, Lab on a chip.

[20]  L. Garrison,et al.  Linking pharmacogenetics-based diagnostics and drugs for personalized medicine. , 2006, Health affairs.

[21]  Niall J. Haslam,et al.  An analysis of the feasibility of short read sequencing , 2005, Nucleic acids research.

[22]  Satoshi Takahashi,et al.  Multiple-sheathflow capillary array DNA analyser , 1993, Nature.

[23]  Timothy B. Stockwell,et al.  The Diploid Genome Sequence of an Individual Human , 2007, PLoS biology.

[24]  R A Mathies,et al.  Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels. , 1999, Analytical chemistry.

[25]  S. Takahashi,et al.  A capillary array gel electrophoresis system using multiple laser focusing for DNA sequencing. , 1996, Analytical chemistry.

[26]  K. A. Wolfe,et al.  Microchip-based purification of DNA from biological samples. , 2003, Analytical chemistry.

[27]  L. Kotler,et al.  DNA sequencing up to 1300 bases in two hours by capillary electrophoresis with mixed replaceable linear polyacrylamide solutions. , 2000, Analytical chemistry.

[28]  James P Landers,et al.  Infrared temperature control system for a completely noncontact polymerase chain reaction in microfluidic chips. , 2007, Analytical chemistry.

[29]  I. Goodhead,et al.  A linear plasmid truncation induces unidirectional flagellar phase change in H:z66 positive Salmonella Typhi , 2007, Molecular microbiology.

[30]  Andreas Graner,et al.  454 sequencing put to the test using the complex genome of barley , 2006, BMC Genomics.

[31]  Jan Berka,et al.  A massively parallel PicoTiterPlate™ based platform for discrete picoliter‐scale polymerase chain reactions , 2003, Electrophoresis.

[32]  James Robinson,et al.  IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex , 2003, Nucleic Acids Res..

[33]  NHGRI SEEKS NEXT GENERATION OF SEQUENCING , 2005, Journal of Investigative Medicine.

[34]  Robert D Schnabel,et al.  SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries , 2008, Nature Methods.

[35]  A. Jeffreys,et al.  Individual-specific ‘fingerprints’ of human DNA , 1985, Nature.

[36]  Joshua M. Korn,et al.  Mapping and sequencing of structural variation from eight human genomes , 2008, Nature.

[37]  J. Lupski,et al.  The complete genome of an individual by massively parallel DNA sequencing , 2008, Nature.

[38]  J. Butler,et al.  Short tandem repeat typing technologies used in human identity testing. , 2007, BioTechniques.

[39]  A. Halpern,et al.  A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Richard A Mathies,et al.  Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Garth D Ehrlich,et al.  Insights into the Genome of Large Sulfur Bacteria Revealed by Analysis of Single Filaments , 2007, PLoS biology.

[42]  Richard A Mathies,et al.  Inline injection microdevice for attomole-scale sanger DNA sequencing. , 2007, Analytical chemistry.

[43]  Tae Seok Seo,et al.  Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[44]  E. Mardis,et al.  What is the future of electrophoresis in large‐scale genomic sequencing? , 2006, Electrophoresis.

[45]  Thomas N. Chiesl,et al.  Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes , 2008, Proceedings of the National Academy of Sciences.

[46]  M. Ronaghi,et al.  A Sequencing Method Based on Real-Time Pyrophosphate , 1998, Science.

[47]  S. Batzoglou,et al.  Whole-Genome Sequencing and Assembly with High-Throughput, Short-Read Technologies , 2007, PloS one.

[48]  R. Mathies,et al.  Monolithic integrated microfluidic DNA amplification and capillary electrophoresis analysis system , 2000 .

[49]  Igor L. Medintz,et al.  Single-molecule DNA amplification and analysis in an integrated microfluidic device. , 2001, Analytical chemistry.

[50]  A. R. Kaiser,et al.  Microfabricated structures for integrated DNA analysis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[51]  R. Fulton,et al.  The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain: evolution during disease progression. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[52]  J. Shendure,et al.  Materials and Methods Som Text Figs. S1 and S2 Tables S1 to S4 References Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome , 2022 .

[53]  Michael G. Roper,et al.  A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability , 2006, Proceedings of the National Academy of Sciences.

[54]  K. A. Wolfe,et al.  Toward a microchip‐based solid‐phase extraction method for isolation of nucleic acids , 2002, Electrophoresis.

[55]  M. Ronaghi,et al.  Real-time DNA sequencing using detection of pyrophosphate release. , 1996, Analytical biochemistry.

[56]  Adrian W. Briggs,et al.  Analysis of one million base pairs of Neanderthal DNA , 2006, Nature.

[57]  E. D. Hyman.  A new method of sequencing DNA. , 1988, Analytical biochemistry.

[58]  Arthur W. Miller,et al.  DNA sequencing of close to 1000 bases in 40 minutes by capillary electrophoresis using dimethyl sulfoxide and urea as denaturants in replaceable linear polyacrylamide solutions , 2002, Electrophoresis.

[59]  Xavier Estivill,et al.  Disorders: Filling the Gaps and Exploring Complexity in Genome-Wide Association Studies , 2022 .

[60]  N. Turro,et al.  Design and synthesis of a photocleavable fluorescent nucleotide 3'-O-allyl-dGTP-PC-Bodipy-FL-510 as a reversible terminator for DNA sequencing by synthesis. , 2006, The Journal of organic chemistry.

[61]  P. Gill,et al.  Encoded evidence: DNA in forensic analysis , 2004, Nature Reviews Genetics.

[62]  M. Ronaghi,et al.  Long-read pyrosequencing using pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer. , 2002, Analytical biochemistry.

[63]  G. Ginsburg,et al.  Prospects for personalized cardiovascular medicine: the impact of genomics. , 2005, Journal of the American College of Cardiology.

[64]  C. Hutchison.  DNA sequencing: bench to bedside and beyond , 2007, Nucleic acids research.

[65]  Christopher J. Easley,et al.  Thermal isolation of microchip reaction chambers for rapid non-contact DNA amplification , 2007 .

[66]  H. John Crabtree,et al.  Construction and evaluation of a capillary array DNA sequencer based on a micromachined sheath‐flow cuvette , 2000, Electrophoresis.

[67]  E. Yeung,et al.  Optimization of Excitation and Detection Geometry for Multiplexed Capillary Array Electrophoresis of DNA Fragments , 1995 .

[68]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[69]  R S Foote,et al.  Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing. , 1998, Analytical chemistry.

[70]  M. Uhlén,et al.  Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay. , 1993, Analytical biochemistry.

[71]  Clive Brown,et al.  Toward the $1000 human genome , 2005 .

[72]  Julia G. Bodmer,et al.  IMGT/HLA Database--a sequence database for the human major histocompatibility complex. , 2001, Nucleic acids research.

[73]  M. Suthanthiran,et al.  Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. , 2001, The New England journal of medicine.

[74]  F. Calafell.  The probability distribution of the number of loci indicating exclusion in a core set of STR markers , 2000, International Journal of Legal Medicine.

[75]  Jay Shendure,et al.  Multiplex amplification of large sets of human exons , 2007, Nature Methods.