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Next-Generation Sequencing and its Applications in RNA-Seq

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A brief introduction in the Next-Generation Sequencing techniques and a short introduction to RNA-Seq.

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Next-Generation Sequencing and its Applications in RNA-Seq

  1. 1. Next-Generation Sequencing and itsApplications in RNA-Seq Perdacher Martin 1
  2. 2. Overview• Next-Generation Sequencing • technologies • applications• RNA-Seq 2
  3. 3. Next-Generation Sequencing technologies• Ion semiconductor sequencing• Pyrosequencing• Sequencing by ligation• Cyclic reversible termination 3
  4. 4. Ion semiconductor sequencing 4
  5. 5. Pyrosequencing• pyrophosphate (PPi) is translated into light emission in two enzymatic steps • ATP-sulfurylase converts PPi in ATP • luciferase converts Luciferin in oxiluciferin and emitted light (ATP is the fuel for this reaction). 5
  6. 6. Sequencing by ligation 6
  7. 7. Cyclic reversible termination• incorporation of modified nucleotides• fluorescence imaging• washing of fluorescent dye and removing of terminating group 7
  8. 8. Overview• Next-Generation Sequencing • technologies • applications• RNA-Seq 8
  9. 9. Anwendungen von Next-Generation Sequencing Transcriptome Sequencing:• Transcriptome sequencing RNA Sequenzierung coding non-coding (rRNA, tRNA, miRNA, siRNA, piRNA)• Genomic sequencing Genomic Sequencing de novo resequencing (copy number variation,• Epigenetic applications SNP point mutations) Epigenetic Chip Seq• Additional applications Methylation profiling Additional: Clip Seq, RIP-Seq, HITS Clip, - RNA interaktion mit RNA-bindenden Proteinen 9
  10. 10. Overview• Next-Generation Sequencing • technologies • applications• RNA-Seq 10
  11. 11. RNA-Seq• biological background• workflow of RNA-Seq• transcriptome reconstruction• differential gene expression 11
  12. 12. background biology of RNA-Seq 12
  13. 13. RNA-Seq• biological background• workflow of RNA-Seq• transcriptome reconstruction• differential gene expression 13
  14. 14. Workflow of RNA-Seq• sample preparation ribo minus and strand specific libs (paired end)• seqeuncing• read mapping Fragmentation of cDNA Purification Adapter ligation Size-based purification of ligation products using PCR of ligation products Purification and sequencing of the fragments 14
  15. 15. Workflow of RNA-Seq• sample preparation ribo minus and strand specific libs (paired end)• seqeuncing• read mapping Fragmentation of cDNA Purification Adapter ligation Size-based purification of ligation products using PCR of ligation products Purification and sequencing of the fragments 14
  16. 16. Read mapping in RNA-Seq 15
  17. 17. Read mapping unspliced alignment match PERFECTLY the• genome unspliced read alignment seed : after mapping, the seed is extended with smith waterman method • seed methods Burrow-Wheeler: transform genome into efficient data structure • Burrows-Wheeler transform methods• spliced aligners • exon first • seed and extend 16
  18. 18. Read mapping (spliced alignment methods)2 steps: unspliced read aligner break reads into shorter (k-mer-)seeds. unmapped reads are split into fragments seed-regions are evaluated and aligned with sensitive alignment independently methods (smith waterman) much slower method pseudogene is dysfunctional and does not contain introns. in the first step of exon-first approaches reads are aligned to the genome based on unspliced read mappers. here it is detected as gene and not as pseudogene. 17
  19. 19. RNA-Seq• biological background• workflow of RNA-Seq• transcriptome reconstruction• differential gene expression 18
  20. 20. Transcriptome reconstruction exon identification: is used for very• genome guided reconstruction short reads (36 bp) first map reads to the genome. the unmapped reads are tested for all • exon identification possibilities of exon-exon junctions not able to identify full transcript structures • genome-guided assembly• genome independent reconstruction 19
  21. 21. Transcriptome reconstruction exon identification: is used for very• genome guided reconstruction short reads (36 bp) first map reads to the genome. the unmapped reads are tested for all • exon identification possibilities of exon-exon junctions not able to identify full transcript structures • genome-guided assembly• genome independent reconstruction 19
  22. 22. Transcriptome reconstruction break reads into k-mers.align reads to the genome build de-brujin graph.and use spliced reads tobuild transcript graph alignment to the genome(node: read fragment, is because of annotationedge: link bet ween purposefragments) one pahtthrough the graphrepresents an isoform ofthe transcript) 20
  23. 23. RNA-Seq• biological background• workflow of RNA-Seq• transcriptome reconstruction• differential gene expression 21
  24. 24. Differential gene expression for RNA-Seq • comparing the count of reads for one gene in different experiments (conditions) • the problem is that one read cannot assigned uniquely to one gene, because of the overlap of genes and their different isoforms 22
  25. 25. Differential gene expression for RNA-Seq 23
  26. 26. Summary• Next-Generation Sequencing, (technologies and applications)• RNA-Seq • biology • workflow of RNA-Seq (read mapping) • transcriptome reconstruction • differential gene expression 24

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