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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-for9456
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1. Kick off meeting for Journal Club on next generation sequencing 4/12/2010
3. 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
4. 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.
5. 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.
7. 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.
9. Comparison of existing methods
10. Usage of sequencing data Transcriptome (RNA) sequencing
Differential expression
Alternative splicing
Complete/targeted genome (DNA) resequencing
Polymorphism and mutation discovery
12. Preprocessing flow Image analysis, base calling
Illuminas 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_(1<k<12) C_k<0.6
Alignment/read-mapping
Illumina pipeline: ELAND aligner.
Bowtie (faster, freely available, Langmead et. al. 2009).
13. Output Count data
14. Literaturesand Meeting Time
15. Propose to meet every 3 weeks.
Meet for one or 1.5 hours each time; have 1-2 presentations.
6 candidate papers.