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Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
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Biotech autumn2012-02-ngs2

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Transcript

  • 1. NGS part 2
  • 2. NGS workflow Создание библиотеки Подготовка субстрата Секвенирование Анализ данных
  • 3. NGS workflow
  • 4. Конструирование библиотек ДНК для приготовления библиотек должна быть фрагментирована - Рестрикция – «коктейль» рестриктаз - УЗ-фрагментация - Без фрагментации
  • 5. Ion Torrent library workflow DNA Shear DNA Fragmented DNA End Polished Fragments Adapter Ligation, Nick-repair & Size Selection PCR AmplifyTemplate Preparation 6
  • 6. Illumina library
  • 7. Ion Torrent vs. Illumina library workflow
  • 8. Nextera DNA sample preparation kit (рекомбиназный способ)
  • 9. Quantification Method Sensitivity E-Gel® System 5 ng / μL Bioanalyzer TM 2100 DNA 1000 chip DNA HS chip 0.1 - 50 ng / μL (50 bp - 7 kb) Qubit® Fluorometer 10 pg /μL - 100 ng /μL qPCR 0.005 ng /μL Library QC - Evaluate library quantity/quality 10
  • 10. Size selection E-Gele Pippin Prep
  • 11. Bioanalyzer (Agilent)
  • 12. Example of gDNA after shearing (good) 13 Example: expected size distribution of fragmented genomic DNA
  • 13. Example of gDNA after shearing (bad) 14 Example: size distribution of oversized fragmented genomic DNA. Troubleshoot: ie fragment for longer time.
  • 14. Example of good DNA Library (end of library prep) 15 Library Example: BioanalyzerTM analysis of good final fragment DNA library (for 100 bp reads). Notice library profile is single, narrow peak.
  • 15. Example of bad DNA Library – with primer dimers 16 Example: library with primer-dimer contamination, resulting in inefficient Template Prep. Re-purify library with AMPure® beads. Unwanted primer- dimers Library
  • 16. Example of bad DNA Library – with concatemers 17 Example: an over-amplified library with concatemer products, resulting in inefficient Template Prep. Re-purify with size-selection and then re-quantify. Unwanted concatemers Library
  • 17. Fragment Shear Pippin Size Selection ___ ___ 18 Shared DNA, Library
  • 18. 19 library insertfwd primer rev primer probe qPCR (real-time PCR)
  • 19. 20 Example qPCR data • E. Coli DH10B Control Library used as Standards in Red (triplicates) • Ion Libraries (triplicates)
  • 20. 21 Example qPCR data
  • 21. Optimizing the library input concentration Amplification Low DNA High DNA Increasing [DNA] “Mixed” readNo read Optimal DNA Too little library input can result in insufficient positive, or “live”, spheres for sequencing Too much library input to template prep can result in too many “mixed” reads 22
  • 22. Microbial sequencing Mitochondrial sequencing Amplicon sequencing • Multiplexed amplicon sequencing for rapid detection of germline and somatic mutations Targeted resequencing by target enrichment RNA-Seq • Whole-transcriptome human RNA • Small RNA Chip-Seq Copy number detection Applications
  • 23. Typical RNA-seq experiment
  • 24. Total RNA sequencing Ligase Enhanced Genome Detection (LEGenD™) technology
  • 25. mRNA-Seq library preparation (ревертазный способ)
  • 26. Small RNA-Seq library preparation (РНК-лигазный способ)
  • 27. Target sequencing Таргетное секвенирование - Целевое секвенирование определенных интересующих участков генома с предварительной наработкой фрагментов и созданием библиотеки – Target enrichment Преимущества: • Увеличение покрытия без увеличения стоимости секвенирования. • Полученные данные гораздо проще обрабатывать и хранить.
  • 28. Target enrichment • PCR – short amplicons – long amlicons • Hybridization – solution phase hybridization – solid phase hybridization
  • 29. Ion PGM Library preparation
  • 30. Ion PGM Library preparation
  • 31. Long amplicons
  • 32. Методы пробоподготовки • ПЦР
  • 33. Ion AmpliSeq •Up to 4,000 primers per pool •One to hundreds of genes •96 barcodes for multiplexing
  • 34. TruSeq Amplicon (Illumina)
  • 35. Fusion PCR
  • 36. Fusion PCR
  • 37. Microfluidic PCR
  • 38. Hybrid capture Solid Solution
  • 39. Solution phase sequence capture using long RNA probes
  • 40. SureSelect (Agilent) TruSeq (Illumina)
  • 41. Solution phase sequence capture using molecular inversion probes (MIP)
  • 42. MIP (molecular inversion probes)
  • 43. HaloPlex Target (Agilent)
  • 44. Solid phase sequence capture using DNA microarrays
  • 45. NimbleGen (Roche)
  • 46. Paired-End sequencing
  • 47. Paired-End sequencing
  • 48. Paired-End sequencing library
  • 49. Detecting structural variants by paired- end mapping
  • 50. Next generation sequencing based approaches to epigenomics • Histone modification profiling (ChIP) • DNA methylation profiling – Enrichment based methods – Bisulfite conversion based methods – Methyl-sensitive restriction based methods – Direct detection
  • 51. ChiP-seq (chromatin immunoprecipitation sequencing)
  • 52. Methylated DNA immuno- precipitation (MeDIP-Seq)
  • 53. Bisulfite sequencing
  • 54. Barcoding
  • 55. Barcoding
  • 56. Barcoding
  • 57. Спасибо за внимание

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