Next-Generation DNA Sequencing Methods

1 ANRV353-GG09-20 ARI25 July 200814:57 Next-Generation DNAS equencing MethodsElaine R. MardisDepartments of Genetics and Molecular Microbiology and Genome Sequencing Center,Washington University School of Medicine, St. Louis MO 63108; email: Rev. Genomics Hum. Genet. :387 402 First published online as a Review in Advance onJune 24, 2008 TheAnnual Review of Genomics and Human Geneticsis online at article s 2008 by Annual rights reserved1527-8204/08/0922-0387$ Wordsmassively parallel Sequencing , Sequencing -by-synthesis, resequencingAbstractRecent scientific discoveries that resulted from the application of Next-Generation DNA Sequencing technologies highlight the striking impactof these massively parallel platforms on genetics.

2 These new meth-ods have expanded previously focused readouts from a variety of DNApreparation protocols to a genome-wide scale and have fine-tuned theirresolution to single base precision. The Sequencing of RNA also hastransitioned and now includes full-length cDNA analyses, serial analysisof gene expression (SAGE)-based Methods , and noncoding RNA dis-covery. Next-Generation Sequencing has also enabled novel applicationssuch as the Sequencing of ancient DNA samples, and has substantiallywidened the scope of metagenomic analysis of environmentally derivedsamples.

3 Taken together, an astounding potential exists for these tech-nologies to bring enormous change in genetic and biological researchand to enhance our fundamental biological here for quick links to Annual Reviews content online, including: Other articles in this volume Top cited articles Top downloaded articles Our comprehensive searchFurtherANNUALREVIEWSAnnu. Rev. Genom. Human Genet. :387-402. Downloaded from Columbia University on 09/03/10. For personal use July 200814:57 INTRODUCTIONThe Sequencing of the reference humangenome was the capstone for many years of hardwork spent developing high-throughput, high-capacity production DNA Sequencing and as-sociated sequence finishing pipelines.

4 The ap-proach used>20,000 large bacterial artificialchromosome (BAC) clones that each containedan approximately 100-kb fragment of the hu-man genome, which together provided an over-lapping set or tiling path through each humanchromosome as determined by physical map-ping (31). In BAC-based Sequencing , each BACclone is amplified in bacterial culture, isolatedin large quantities, and sheared to produce size-selected pieces of approximately 2 3 kb. Thesepieces are subcloned into plasmid vectors, am-plified in bacterial culture, and the DNA isselectively isolated prior to Sequencing .

5 Bygenerating approximately eightfold oversam-pling (coverage) of each BAC clone in plasmidsubclone equivalents, computer-aided assemblycan largely recreate the BAC insert sequencein contigs (contiguous stretches of assembledsequence reads). Subsequent refinement, in-cluding gap closure and sequence qualityimprovement (finishing), produces a single con-tiguous stretch of high-quality sequence (typi-cally with less than 1 error per 40,000 bases).Since the completion of the human genomeproject (HGP) (26, 51), substantive changeshave occurred in the approach to genome se-quencing that have moved away from BAC-based approaches and toward whole-genomesequencing (WGS), with changes in the ac-companying assembly algorithms.

6 In the WGSapproach, the genomic DNA is sheared di-rectly into several distinct size classes and placedinto plasmid and fosmid subclones. Oversam-pling the ends of these subclones to gener-ate paired-end Sequencing reads provides thenecessary linking information to fuel whole-genome assembly algorithms. The net result isthat genomes can be sequenced more rapidlyand more readily, but highly polymorphic orhighly repetitive genomes remain quite frag-mented after these dramatic changes in sequenc-ing and assembly approaches, the primary dataproduction for most genome Sequencing sincethe HGP has relied on the same type of capillarysequencing instruments as for the HGP.

7 How-ever, that scenario is rapidly changing owingto the invention and commercial introductionof several revolutionary approaches to DNAsequencing, the so-called Next-Generation se-quencing technologies. Although these instru-ments only began to become commerciallyavailable in 2004, they already are having a ma-jor impact on our ability to explore and an-swer genome-wide biological questions; morethan 100 Next-Generation Sequencing relatedmanuscripts have appeared to date in the peer-reviewed literature.

8 These technologies are notonly changing our genome Sequencing ap-proaches and the associated timelines and costs,but also accelerating and altering a wide va-riety of types of biological inquiry that havehistorically used a Sequencing -based readout,or effecting a transition to this type of read-out, as detailed in this review. Furthermore, Next-Generation platforms are helping to openentirely new areas of biological inquiry, includ-ing the investigation of ancient genomes, thecharacterization of ecological diversity, and theidentification of unknown etiologic DNASEQUENCINGT hree platforms for massively parallel DNAsequencing read production are in reasonablywidespread use at present.

9 The Roche/454 FLX (30) ( ), the Illumina/Solexa Genome Analyzer (7) ( ), and theApplied Biosystems SOLiDTMS ystem ( / SolidKnowledge / flash / 102207 ). Recently, another two massivelyparallelsystemswereannounced:th eHelicos HeliscopeTM( )andPacificBiosciencesSMRT( ) instruments. The388 MardisAnnu. Rev. Genom. Human Genet. :387-402. Downloaded from Columbia University on 09/03/10. For personal use July 200814:57 Helicos system only recently became com-mercially available, and the Pacific Biosciencesinstrument will likely launch commerciallyin early 2010.

10 Each platform embodies acomplex interplay of enzymology, chemistry,high-resolution optics, hardware, and softwareengineering. These instruments allow highlystreamlined sample preparation steps prior toDNA Sequencing , which provides a significanttime savings and a minimal requirementfor associated equipment in comparison tothe highly automated, multistep pipelinesnecessary for clone-based high-throughputsequencing. By different approaches outlinedbelow, each technology seeks to amplify singlestrands of a fragment library and performsequencing reactions on the amplified fragment libraries are obtained by anneal-ing platform-specific linkers to blunt-endedfragments generated directly from a genome orDNA source of interest.