Biotech autumn2012-02-ngs2

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Biotech autumn2012-02-ngs2

  1. 1. NGS part 2
  2. 2. NGS workflow Создание библиотеки Подготовка субстрата Секвенирование Анализ данных
  3. 3. NGS workflow
  4. 4. Конструирование библиотек ДНК для приготовления библиотек должна быть фрагментирована - Рестрикция – «коктейль» рестриктаз - УЗ-фрагментация - Без фрагментации
  5. 5. Ion Torrent library workflow DNA Shear DNA Fragmented DNA End Polished Fragments Adapter Ligation, Nick-repair & Size Selection PCR AmplifyTemplate Preparation 6
  6. 6. Illumina library
  7. 7. Ion Torrent vs. Illumina library workflow
  8. 8. Nextera DNA sample preparation kit (рекомбиназный способ)
  9. 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. 10. Size selection E-Gele Pippin Prep
  11. 11. Bioanalyzer (Agilent)
  12. 12. Example of gDNA after shearing (good) 13 Example: expected size distribution of fragmented genomic DNA
  13. 13. Example of gDNA after shearing (bad) 14 Example: size distribution of oversized fragmented genomic DNA. Troubleshoot: ie fragment for longer time.
  14. 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. 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. 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. 17. Fragment Shear Pippin Size Selection ___ ___ 18 Shared DNA, Library
  18. 18. 19 library insertfwd primer rev primer probe qPCR (real-time PCR)
  19. 19. 20 Example qPCR data • E. Coli DH10B Control Library used as Standards in Red (triplicates) • Ion Libraries (triplicates)
  20. 20. 21 Example qPCR data
  21. 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. 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. 23. Typical RNA-seq experiment
  24. 24. Total RNA sequencing Ligase Enhanced Genome Detection (LEGenD™) technology
  25. 25. mRNA-Seq library preparation (ревертазный способ)
  26. 26. Small RNA-Seq library preparation (РНК-лигазный способ)
  27. 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. 28. Target enrichment • PCR – short amplicons – long amlicons • Hybridization – solution phase hybridization – solid phase hybridization
  29. 29. Ion PGM Library preparation
  30. 30. Ion PGM Library preparation
  31. 31. Long amplicons
  32. 32. Методы пробоподготовки • ПЦР
  33. 33. Ion AmpliSeq •Up to 4,000 primers per pool •One to hundreds of genes •96 barcodes for multiplexing
  34. 34. TruSeq Amplicon (Illumina)
  35. 35. Fusion PCR
  36. 36. Fusion PCR
  37. 37. Microfluidic PCR
  38. 38. Hybrid capture Solid Solution
  39. 39. Solution phase sequence capture using long RNA probes
  40. 40. SureSelect (Agilent) TruSeq (Illumina)
  41. 41. Solution phase sequence capture using molecular inversion probes (MIP)
  42. 42. MIP (molecular inversion probes)
  43. 43. HaloPlex Target (Agilent)
  44. 44. Solid phase sequence capture using DNA microarrays
  45. 45. NimbleGen (Roche)
  46. 46. Paired-End sequencing
  47. 47. Paired-End sequencing
  48. 48. Paired-End sequencing library
  49. 49. Detecting structural variants by paired- end mapping
  50. 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. 51. ChiP-seq (chromatin immunoprecipitation sequencing)
  52. 52. Methylated DNA immuno- precipitation (MeDIP-Seq)
  53. 53. Bisulfite sequencing
  54. 54. Barcoding
  55. 55. Barcoding
  56. 56. Barcoding
  57. 57. Спасибо за внимание

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