Introduction_Journal_Club.pptVIP

Outline A brief introduction to the next generation sequencing technology/platform (20 minutes). Literature papers. Decide the time/frequency of the club. Introduction Terminology: Ligase: An enzyme that links two large molecules by forming a new chemical bond. DNA ligase: A special type of ligase that can link together two DNA strands that have double-strand break, or join the ends on only one of the two strand. Polymerase: Can add free nucleotides to only the 3’ end of the newly-forming strand. This results in elongation of the new strand in a 5’-3’ direction. PCR: polymerase chain reaction Next Generation Sequencing DNA is fragmented Adaptors ligated to fragments Several possible protocols yield array of PCR colonies. Emulsion PCR Bridge PCR Enyzmatic extension with fluorescently tagged nucleotides. Cyclic readout by imaging the array. Emulsion PCR Fragments, with adaptors, are PCR amplified within a water drop in oil. One primer is attached to the surface of a bead. Used by 454, Polonator and SOLiD. Bridge PCR DNA fragments are flanked with adaptors. A flat surface coated with two types of primers, corresponding to the adaptors. Amplification proceeds in cycles, with one end of each bridge tethered to the surface. Used by Solexa. Comparison of existing methods Usage of sequencing data Transcriptome (RNA) sequencing Differential expression Alternative splicing Complete/targeted genome (DNA) resequencing Polymorphism and mutation discovery Preprocessing flow Image analysis, base calling Illumina’s pipeline: Firecrest image analysis Bustard base-calling: provides read quality measures Chastity at circle k: (max_A+max_C+max_G+max_T)/[I(1)+I(2)] Purity filter (PF): min_(1k12) C_k0.6 Alignment/read-mapping Illumina pipeline: ELAND aligner. Bowtie (faster, freely available, Langmead et. al. 2009). Literaturesand Meeting Time Propose to meet every 3 weeks. Meet for one or 1.5 hours each time; have 1-2 presentations. 6 candidate papers. * Kick off