Special Topics in GenomicsWork flow of conventional versus second-generation sequencingAvailable next-generation sequencing platformsExample: Illumina/SolexaIllumina/SolexaClonal amplification of sequencing features in the second-generation sequencingStrategies for cyclic array sequencingConventional sequencingSecond-generation DNA sequencing technologiesApplications of next-generation sequencingBase callingBase calling: Alta-CyclicAlta-Cyclic performanceChIP-SeqChIP-Seq AnalysisAlignmentPeak detectionTwo common designsOne sample analysisThe constant rate assumption does not hold!FDR estimation based on Poisson and negative binomial modelRead direction provides extra informationCisGenome procedureTwo sample analysisCisGenome two sample analysisA comparative study of ChIP-chip and ChIP-seqIntersectionSignal correlationVisual comparisonComparison of peak detection resultsAre array specific peaks noise or signal?Effects of read number in ChIP-seqRNA-SeqReproducibility, linearity and sensitivityERANGESummarySpecial Topics in GenomicsNext-generation SequencingWork flow of conventional versus second-generation sequencing(a) With high-throughput shotgun Sanger sequencing, genomic DNA is fragmented, then cloned to a plasmid vector and used to transform E. coli. For each sequencing reaction, a single bacterial colony is picked and plasmid DNA isolated. Each cycle sequencing reaction takes place within a microliter-scale volume, generating a ladder of ddNTP-terminated, dye-labeled products, which are subjected to high-resolution electrophoretic separation within one of 96 or 384 capillaries in one run of a sequencing instrument. As fluorescently labeled fragments of discrete sizes pass a detector, the four-channel emission spectrum is used to generate a sequencing trace. (b) In shotgun sequencing with cyclic-array methods, common adaptors are ligated to fragmented genomic DNA, which is then subjected to one of several protocols that results in an array of millions of spatially immobilized PCR colonies or 'polonies'15. Each polony consists of many copies of a single shotgun library fragment. As all polonies are tethered to a planar array, a single microliter-scale reagent volume (e.g., for primer hybridization and then for enzymatic extension reactions) can be applied to manipulate all array features in parallel. Similarly, imaging-based detection of fluorescent labels incorporated with each extension can be used to acquire sequencing data on all features in parallel. Successive iterations of enzymatic interrogation and imaging are used to build up a contiguous sequencing read for each array feature.Jay Shendure & Hanlee Ji, Nature Biotechnology 26, 1135 - 1145 (2008)Available next-generation sequencing platforms•Illumina/Solexa•ABI SOLiD•Roche 454•Polonator•HeliScope•…Example: Illumina/Solexa 1. Prepare genomic DNA 2. Attach DNA to surface 3. Bridge amplification 4. Fragement become double stranded5. Denature the double stranded molecules6. Complete amplificationIllumina/Solexa 7. Determine first base8. Image first base9. Determine second base10. Image second base11. Sequence reads over multiple cycles12. Align data. >50 milliion clusters/flow cell, each 1000 copies of the same template, 1 billion bases per run, 1% of the cost of capillary-based method. (From: http://www.illumina.com/downloads/SS_DNAsequencing.pdf)Clonal amplification of sequencing features in the second-generation sequencing(a) The 454, the Polonator and SOLiD platforms rely on emulsion PCR20 to amplify clonal sequencing features. In brief, an in vitro–constructed adaptor-flanked shotgun library (shown as gold and turquoise adaptors flanking unique inserts) is PCR amplified (that is, multi-template PCR, not multiplex PCR, as only a single primer pair is used, corresponding to the gold and turquoise adaptors) in the context of a water-in-oil emulsion. One of the PCR primers is tethered to the surface (5'-attached) of micron-scale beads that are also included in the reaction. A low template concentration results in most bead-containing compartments having either zero or one template molecule present. In productive emulsion compartments (where both a bead and template molecule is present), PCR amplicons are captured to the surface of the bead. After breaking the emulsion, beads bearing amplification products can be selectively enriched. Each clonally amplified bead will bear on its surface PCR products corresponding to amplification of a single molecule from the template library. (b) The Solexa technology relies on bridge PCR21, 22 (aka 'cluster PCR') to amplify clonal sequencing features. In brief, an in vitro–constructed adaptor-flanked shotgun library is PCR amplified, but both primers densely coat the surface of a solid substrate, attached at their 5' ends by a flexible linker. As a consequence, amplification products originating from any given member of the template library remain locally tethered near the point of origin. At the conclusion of the PCR, each clonal cluster contains 1,000 copies of a single member of the template library. Accurate measurement of the concentration of the template library is critical to maximize the cluster density while simultaneously avoiding overcrowding.Jay Shendure & Hanlee Ji, Nature Biotechnology 26, 1135 - 1145 (2008)Strategies for cyclic array sequencing(a) With the 454 platform, clonally amplified 28-m beads generated by emulsion PCR serve as sequencing features and are randomly deposited to a microfabricated array of picoliter-scale wells. With pyrosequencing, each cycle consists of the introduction of a single nucleotide species, followed by addition of substrate (luciferin, adenosine 5'-phosphosulphate) to drive light production at wells where polymerase-driven incorporation of that nucleotide took place. This is followed by an apyrase wash to remove unincorporated nucleotide. (b) With the Solexa technology, a dense array of clonally amplified sequencing features is generated directly on a surface by bridge PCR. Each sequencing cycle includes the simultaneous addition of a mixture of four modified deoxynucleotide species, each bearing one of four fluorescent labels and a reversibly terminating moiety at the 3' hydroxyl position. A modified DNA polymerase drives synchronous extension of primed sequencing features. This is followed by imaging in four channels and then cleavage of both the fluorescent labels and the terminating moiety. (c) With the SOLiD