Prepare adapter ligated ssDNA library

Next Generation Sequencing Platforms – Sequencing by synthesis (SBS) • 454/Pyrosequencing • Illumina/Solexa • Helicos • Pacbio • (Charge-based detection system, Now-sequencing) – Sequencing by hybridization • SOLiD Pyrosequencing: Sequencing-By-Synthesis [...]... different library adapters (total of 12 adapters) – Each MID library adapter has an added, specially encoded 10-base region – Used to “bar-code” up to 12 different genomic library samples to be run in the same region of a single sequencing run Standard Library Seq primer #bases: MID Library Read Primer A 40 Key 4 Library fragment Primer B Seq primer Read Primer A #bases: 15 Key MID 1 4 10 Library fragment... Sequencing Center Transcriptome Analysis Workflow Comparison GS FLX (clonal sequencing ensured through emPCR) emPCR cDNA libraries (short tag library, EST library) Time: Days Sequencing Sanger (E coli cloning, often concatemerization) cDNA libraries (short tag library, EST library) Concatemerization, insert fragments into vectors and clone into bacteria CSB2008 August 2008 Sequencing Grow, pick colonies Template... and ligation rounds CSB2008 August 2008 UCSC Sequencing Center Library Construction 2 different libraries can depending on the application and desired information CSB2008 August 2008 UCSC Sequencing Center Fragment Library DNA is fragmented and PCR primer adaptors are ligated to the DNA CSB2008 August 2008 UCSC Sequencing Center Mate-Pair Library DNA is sheared, selected for a desired input size, and... Example • 19 Kb region from Chromosome 4 Targeted Exons Seq-Cap Array Probes GS FLX Seq Reads Sequencing Coverage CSB2008 August 2008 UCSC Sequencing Center SOLiD Library Preparation The SOLiD system uses either a fragment library or a mate-paired library depending on the user’s desired information or application CSB2008 August 2008 UCSC Sequencing Center Emulsion PCR and Bead Enrichment PCR takes place... microreactors DNA library preparation and titration 4.5 h and 10.5 h CSB2008 August 2008 Clonal amplification occurs inside microreactors emPCR 8.0 h Break microreactors , enrich for DNA-positive beads Sequencin g 7.5 h UCSC Sequencing Center Sequencing Workflow Loading of PicoTiterPlate Device Depositing DNA beads into the PicoTiterPlate device • Well diameter: average of 44 µm • • Amplified sstDNA library. .. Library Seq primer #bases: MID Library Read Primer A 40 Key 4 Library fragment Primer B Seq primer Read Primer A #bases: 15 Key MID 1 4 10 Library fragment Primer B Primer A Key MID 2 Library fragment Primer B Primer A Key MID n Library fragment Primer B CSB2008 August 2008 UCSC Sequencing Center Paired-End Applications • ~100 bp sequencing tags separated by 3 kb spacing • Use for de novo assembly – Order... nucleotide complementary to the template strand generates a light signal The light signal is recorded by the CCD camera The signal strength is proportional to the number of nucleotides incorporated DNA library preparation and titration 4.5 h and 10.5 h CSB2008 August 2008 emPCR 8.0 h Key sequence Flowgram Sequencin g 7.5 h UCSC Sequencing Center GS FLX Data Analysis Flowgram Generation 4mer T A C G 3-mer . sequencing Small RNA CSB2008 August 2008 UCSC Sequencing Center 1) Prepare Adapter Ligated ssDNA Library (A-[insert]-B) 2) EmPCR: Clonal Amplification on 28 µ. Center Sequencing Workflow Library Preparation 8.0 h 7.5 h 4.5 h and 10.5 h DNA library preparation and titration emPCR Sequencin g sstDNA library created with