Illumina Sequencing Overview

Transcription

1 Illumina Sequencing Overview Part # _Rev.C 2013 Illumina, Inc. All rights reserved. Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cbot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iselect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa, TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

2 Session Objectives By the end of this training, you will be able to: List the major steps in the Illumina sequencing workflow Describe cluster generation Discuss the sequencing by synthesis process 2 Part # _Rev.D FOR RESEARCH USE ONLY

3 Illumina Sequencing Workflow Library Preparation Cluster Generation Sequencing Data Analysis 3 Part # _Rev.D FOR RESEARCH USE ONLY

4 Illumina Sequencing Workflow Library Preparation Cluster Generation Sequencing Data Analysis 4 Part # _Rev.D FOR RESEARCH USE ONLY

5 Library Prep is Critical for Successful Sequencing Dual Index Library shown The aim of library prep is to obtain nucleic acid fragments with adapters attached on both ends 5 Part # _Rev.D FOR RESEARCH USE ONLY

6 Illumina Sequencing Workflow Library Preparation Cluster Generation Sequencing Data Analysis 6 Part # _Rev.D FOR RESEARCH USE ONLY

7 What is a Flow Cell? Cluster generation occurs on a flow cell A flow cell is a thick glass slide with channels or lanes Each lane is randomly coated with a lawn of oligos that are complementary to library adapters 7 Part # _Rev.D FOR RESEARCH USE ONLY

8 What is a Cluster? Clusters are bright spots on an image Each cluster represents thousands of copies of the same DNA strand in a 1 2 micron spot Single DNA Library Cycle 1, G Channel Amplified Clonal Cluster 8 Part # _Rev.D FOR RESEARCH USE ONLY

9 Instrumentation Single DNA Library Amplified Clonal Cluster cbot Sequencer 9 Part # _Rev.D FOR RESEARCH USE ONLY

10 Hybridize Fragment & Extend Single DNA libraries are hybridized to primer lawn Adapter sequence Bound libraries are then extended by polymerases Surface of flow cell coated with a lawn of oligo pairs 3 extension 10 Part # _Rev.D FOR RESEARCH USE ONLY

11 Denature Double-Stranded DNA Double-stranded molecule is denatured Original template Newly synthesized strand Original template washed away discard Newly synthesized strand is covalently attached to flow cell surface 11 Part # _Rev.D FOR RESEARCH USE ONLY

12 Single-Stranded DNA NOTE: Single molecules bind to flow cell in a random pattern 12 Part # _Rev.D FOR RESEARCH USE ONLY

13 Bridge Amplification Single-stranded molecule flips over and forms a bridge by hybridizing to adjacent, complementary primer Hybridized primer is extended by polymerases 13 Part # _Rev.D FOR RESEARCH USE ONLY

14 Bridge Amplification Double-stranded bridge is formed 14 Part # _Rev.D FOR RESEARCH USE ONLY

15 Denature Double-Stranded Bridge Double-stranded bridge is denatured Result: Two copies of covalently bound single-stranded templates 15 Part # _Rev.D FOR RESEARCH USE ONLY

16 Bridge Amplification Single-stranded molecules flip over to hybridize to adjacent primers Hybridized primer is extended by polymerase 16 Part # _Rev.D FOR RESEARCH USE ONLY

17 Bridge Amplification Bridge amplification cycle is repeated until multiple bridges are formed 17 Part # _Rev.D FOR RESEARCH USE ONLY

18 Linearization dsdna bridges are denatured 18 Part # _Rev.D FOR RESEARCH USE ONLY

19 Reverse Strand Cleavage Reverse strands are cleaved and washed away, leaving a cluster with forward strands only 19 Part # _Rev.D FOR RESEARCH USE ONLY

20 Blocking Free 3 ends are blocked to prevent unwanted DNA priming 20 Part # _Rev.D FOR RESEARCH USE ONLY

21 Read 1 Primer Hybridization Sequencing primer is hybridized to adapter sequence Sequencing primer 21 Part # _Rev.D FOR RESEARCH USE ONLY

22 Illumina Sequencing Workflow Library Preparation Cluster Generation Sequencing Data Analysis 22 Part # _Rev.D FOR RESEARCH USE ONLY

23 Sequencing By Synthesis (SBS) Add 4 Fl-NTP s + Polymerase Incorporated FI- NTP imaged Terminator & fluorescent dye cleaved from FI-NTP X Part # _Rev.D FOR RESEARCH USE ONLY

24 Four Channel SBS Chemistry: GA, HiSeq, MiSeq Each of the four DNA bases emit an intensity of a unique wavelength Collects four images: During each cycle, each cluster appears in only one of four images 26 Part # _Rev.D FOR RESEARCH USE ONLY

25 Two Channel SBS NextSeq 500 Two channel SBS uses two images Clusters appearing in green only are T Clusters appearing in red only are C Clusters appearing in both images are A Clusters not present in either green nor red are G Cluster intensities are plotted and bases are called accordingly 27 Part # _Rev.D FOR RESEARCH USE ONLY

26 Paired-End Sequencing 28 Part # _Rev.D FOR RESEARCH USE ONLY

27 Paired-End Sequencing Sequenced strand is stripped off Blocked 3 -ends Sequenced strand 3 -ends of template strands and lawn primers are unblocked 30 Part # _Rev.D FOR RESEARCH USE ONLY

28 Paired-End Sequencing Single-stranded template loops over to form a bridge by hybridizing with a lawn primer 3 -ends of lawn primer are extended Bridge formation 3 extension 31 Part # _Rev.D FOR RESEARCH USE ONLY

29 Paired-End Sequencing Double stranded DNA 32 Part # _Rev.D FOR RESEARCH USE ONLY

30 Paired-End Sequencing Bridges are linearized and the original forward template is cleaved Original forward strand 33 Part # _Rev.D FOR RESEARCH USE ONLY

31 Paired-End Sequencing Free 3 ends of the reverse template and lawn primers are blocked to prevent unwanted DNA priming Blocked 3 -ends Sequencing primer Sequencing primer is hybridized to adapter sequence Reverse strand template 34 Part # _Rev.D FOR RESEARCH USE ONLY

32 Sequencing By Synthesis 2 nd Read Add 4 Fl-NTP s + Polymerase Incorporated FI- NTP imaged Terminator & fluorescent dye cleaved from FI-NTP X Part # _Rev.D FOR RESEARCH USE ONLY

33 Illumina Sequencing Workflow Library Preparation Cluster Generation Sequencing Data Analysis 40 Part # _Rev.D FOR RESEARCH USE ONLY

34 Data Analysis Overview Control Software Analysis Software Visualization Software 41 Part # _Rev.D FOR RESEARCH USE ONLY

35 Analysis Overview Analysis Type Software Outputs Control Software ICS/MCS/RTA Images, Intensities and Base Calls Analysis Software CASAVA HiSeq Analysis Software MiSeq Reporter Alignments, Variant Detection Visualization Software Annotation, Filtering, Reports 42 Part # _Rev.D FOR RESEARCH USE ONLY

36 Summary Library Preparation Cluster Generation Sequencing Data Analysis 43 Part # _Rev.D FOR RESEARCH USE ONLY

37 Questions? 44 Part # _Rev.D FOR RESEARCH USE ONLY