Lecture 16 sequencing technologies

BMMB 554 2022 Lecture 16

Transcript

1. L4: SEQUENCING TECHNOLOGIES Foundations in Data Driven Life Sciences BMMB 554
2. Today’s learning objective • Understand how DNA sequencing works • Understand the relative advantages of current sequencing platforms.
3. Sanger sequencing
4. Sanger sequencing 5’-ACGGT-3’ ||||| 3’-TGCCA-5’ DNA to sequence Other ingredients: • DNA polymerase • dNTPs: • A • C • G • T • Labeled ddNTPs: • A* • C* • G* • T* ACGGT ACGGT ACGGT ACGGT ACGGT PCR & denature ssDNA
5. Sanger sequencing ACGGT | T ACGGT | T ACGGT | T* ACGGT | T ACGGT | T ACGGT | T ACGGT | T* DNA pol base 1
6. Sanger sequencing ACGGT || TG ACGGT || TG ACGGT | T* ACGGT || TG* ACGGT || TG ACGGT || TG ACGGT | T* DNA pol base 2
7. Sanger sequencing ACGGT ||| TGC* ACGGT ||| TGC ACGGT | T* ACGGT || TG* ACGGT ||| TGC ACGGT ||| TGC ACGGT | T* DNA pol base 3
8. Sanger sequencing ACGGT ||| TGC* ACGGT |||| TGCC* ACGGT | T* ACGGT || TG* ACGGT |||| TGCC* ACGGT |||| TGCC ACGGT | T* DNA pol base 4
9. Sanger sequencing ACGGT ||| TGC* ACGGT |||| TGCC* ACGGT | T* ACGGT || TG* ACGGT |||| TGCC* ACGGT ||||| TGCCA* ACGGT | T* DNA pol base 5
10. Sanger sequencing ACGGT ||| TGC* ACGGT |||| TGCC* ACGGT | T* ACGGT || TG* ACGGT |||| TGCC* ACGGT ||||| TGCCA* ACGGT | T* DNA pol base 5
11. Sanger sequencing Electrophoresis (sort by size) Excite with laser ACGGT | T* ACGGT | T* ACGGT || TG* ACGGT ||| TGC* ACGGT |||| TGCC* ACGGT |||| TGCC* ACGGT ||||| TGCCA* A C C G T
12. Sanger sequencing
13. Sanger sequencing • Read length: Up to 1,000bp • Accuracy: 99.99% • Throughput: 96 reads (capillary machine) • Time: 2 hours • Run cost: $1000/Mbp • Machine cost: $300K • Most early whole-genome sequencing projects used Sanger sequencing with a ‘shotgun’ approach. • Useful for closing gaps in sequence assemblies or for sequencing defined positions on the genome.
14. Illumina (sequencing by synthesis) Flow-cell More details: http://seqanswers.com/forums/showthread.php?t=21
15. Prepare Genomic DNA Sample • Fragment DNA of interest • Ligate sequencing adapters • Denature dsDNA into ssDNA Image retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf Slide credits: USD Bioinformatics
16. Attach DNA to Surface • Flow cell is coated with primers complementary to sequencing adapters • ssDNA attaches to surface of flow cell Images retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf Flow Cell
17. Bridge Amplification • Unlabeled nucleotides and polymerase enzyme are added to initiate bridge amplification Image retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf
18. Fragments Become Double Stranded Image retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf
19. Denature the Double Stranded Molecules • Each dsDNA results in two ssDNA molecules, in opposite orientations Image retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf
20. Steps 5-7 Repeats • Cycles of new strand synthesis and denaturation to make multiple copies of the same sequence (amplification) Image retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf
21. Bridge amplification Image source: wikipedia
22. Determine First Base • Add sequencing reagents • Primers (single orientation) • Polymerase • Fluorescently labeled nucleotides • Buffer • First base incorporated Image retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf
23. Image First Base • Remove unincorporated bases • Detect fluorescent signal • Deblock and remove the fluorescent signal à new cycle Image retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf Image retrieved from http://research.stowers- institute.org/microscopy/external/PowerpointPresentations/ppt/Methods_Technology/ KSH_Tech&Methods_012808Final.pdf
24. Determine Second Base • Add sequencing reagents • Primers • Polymerase • Fluorescently labeled nucleotides • Buffer • Second base incorporated Image retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf
25. Image Second Chemistry Cycle • Remove unincorporated bases • Detect Signal • Deblock and remove the fluorescent signal à new cycle Image retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf
26. Sequence Reads Over Multiple Chemistry Cycles • The identity of each base of a cluster is read off from sequential images Image retrieved from http://res.illumina.com/documents/products/techsp otlights/techspotlight_sequencing.pdf Image retrieved from http://research.stowers- institute.org/microscopy/external/PowerpointPresentations/ppt/Methods_Technology/KSH_Tech&Met hods_012808Final.pdf
27. Illumina HiSeq • Read length: up to 150bp (paired-end possible) • Accuracy: 99% • Throughput: ~300,000,000 reads x 8 lanes • Time: 3-4 days per run • Run cost: $0.03/Mbp • Machine cost: $600K
28. Illumina NextSeq • Read length: up to 150bp (paired-end possible) • Accuracy: 99% • Throughput: ~300,000,000 reads • Time: 1 day per run • Run cost: $0.03/Mbp • Machine cost: $250K
29. Pacific Biosciences SMRT sequencing • https://youtu.be/WMZmG00uhwU
30. PacBio • Figure 1 from Flushberg et al. 2010.
31. PacBio • One of the major drawbacks of SNS technologies is a relatively high error. In the case of PacBio it is somewhere between 10 to 25%. However, because PacBio uses library produced by ligating bell-shaped adapters to DNA molecules, a single circular molecule can be sequenced multiple times allowing for error correction (Figure 1a from Wenger et al. 2019.) • Note that it is also possible to read a longer insert just generating what is called Continuous Long Reads (CLRs). These are obviously much longer but are less accurate. Thus current PacBio systems produce two types of reads: Circular Consensus Reads (CCR) and Continuous Long Reads (CLRs). A subset of high quality CCR reads (with base Q>20Q>20) is called HiFi reads.
32. Pacific Biosciences Sequel • Read length: 10 – 15Kbp • Accuracy: ~90% • Throughput: ~500,000 reads • Time: 240 minutes • Run cost: $0.04/Mbp • Machine cost: $350K
33. Oxford Nanopore As DNA passes through the nanopore, different bases can be detected as changes in resistance properties. Potential to sequence entire chromosomes at once. https://www.youtube.com/watch?v=GUb1TZvMWsw
34. Oxford Nanopore MinION SmidgION
35. Oxford Nanopore MinION • Read length: >100Kbp • Accuracy: ~85-95% • Throughput: 30Gbp • Cost: $1000 (starter pack with 2 cells)
36. Summary • Current DNA sequencing platforms produce hundreds of millions of short sequence reads at low cost. • Sequencing platforms report on their confidence in their base calls.