Bloomberg School BIO 624 - Topics in Genomics (36 pages)

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Topics in Genomics



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Topics in Genomics

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Pages:
36
School:
Johns Hopkins Bloomberg School of Public Health
Course:
Bio 624 - Foundations of Biomedical Physiology
Foundations of Biomedical Physiology Documents

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Special Topics in Genomics Next generation Sequencing Work flow of conventional versus secondgeneration 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 microliterscale reagent volume e g for primer hybridization and then for enzymatic extension reactions can be applied to manipulate all array features in parallel Similarly imagingbased 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 stranded 5 Denature the double stranded molecules 6 Complete amplification Illumina Solexa 7 Determine first base 8 Image first



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