Presentation Transcript

1. High-Throughput Sequencing Technologies

   Biological Sequence Analysis BNFO 691/602 Spring 2014 Mark Reimers
2. Outline What can we do with next-generation sequencing? De novo sequencing of simple genomes Re-sequence individual variations Generate genome-wide quantitative data for a variety of assays What technologies are now available and which are up-and-coming? Roche, Illumina, SOLiD, Ion Torrent, etc…
3. What is High-Throughput Sequencing? Generating many thousands or millions of short (30 to 1,000 base) sequences by sequencing parts of longer (200+ base) DNA fragments Most research uses reads from one end of a fragment (single-end), but most technologies can be adapted to make paired-end reads on opposite strands
4. Full Genome Re-sequencing has been done for many cancers and rare clinical disorders
5. Exomesequencing is a cost-effective to identify denovo protein coding mutations
6. Targeted re-sequencing of a few relevant genes can identify diverse critical mutations across a large number of cases
7. RNA-seq
8. ChIP-seq
9. DNA methylation profiling mC  C C  U After PCR C  C U  T PCR+Seq
10. DNAse Hyper-sensitivity DNAse I enzyme cuts DNA Much more likely to cut at open chromatin Two approaches: Cut slowly then fragment and sequence ends Cut rapidly then sequence short fragments
11. Mapping of chromatin interactions (5C) (courtesy Elemento lab)
12. HTS Technologies Roche-454 (will close 2016) Illumina SOLiD Ion Torrent Newer Technologies Outlook
13. Founded by Jonathan Rothberg as a secret project (code-named ‘454’) within CuraGen
14. Roche 454 Sequencing Metzker, NG 2010
15. Roche 454 Sequencing
16. Roche 454 Peak Heights Data
17. Advantages & Drawbacks PRO Long reads are uniquely identifiable Relatively quick ~20 hours total CON Cost is relatively high Frequent errors in runs of bases Frequent G-A transitions
18. Best Uses of Roche 454 De novo small genome (prokaryote or small eukaryote genome) sequencing Metagenomics by16S profiling Used to be best for metagenomics by random sequencing new long reads from Illumina are competitive Targeted re-sequencing of small samples
19. Illumina (Solexa) Genome Analyzer and Flow Cell
20. IlluminaOn-Chip Amplification
21. Illumina (Solexa) Sequencing
22. Paired-End Illumina Method Paired-end reads are easy on Illumina because the clusters are generated by ligated linkers. Different linkers and primers are attached to each end
23. Advantages & Drawbacks PRO Very high throughput Most widespread technology so that comparisons seem easier CON Sequencing representation biases, especially at beginning Slow – up to a week for a run
24. Best Uses of Illumina Expression analysis (RNA-Seq) Chromatin Immunoprecipitation (ChIP-Seq) Metagenomics by random sequencing
25. SOLiDSequencing by Oligonucleotide Ligation and Detection
26. SOLiD History George Church licensed his ‘polony’ technique to Agencourt Personal Genomics ABI acquired the SOLiD technology from Agencourt in 2006
27. SOLiD Preparation Steps Prepare either single or ‘mate-pair’ library from DNA fragments Attach library molecules to beads; amplify library by emulsion PCR Modify 3’ ends of clones; attach beads to surface
28. Emulsion PCR Emulsion PCR isolates individual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase. A polymerase chain reaction (PCR) then coats each bead with clonal copies of the DNA molecule. The bead is immobilized for sequencing.
29. ABI SOLiD Sequencing Cycle
30. SOLiD Reads Each Base Twice Most bases are matched by two primers in different ligation cycles
31. SOLiD Color Coding Scheme Blue is color of homopolymer runs If you translate color reads directly into base reads then every sequence with an error in the color calls will result in a frame-shift of the base calls. it is best to convert the reference sequence into color-space. There is one unambiguous conversion of a base reference sequence into color-space, but there are four possible conversions of a color string into base strings
32. Advantages & Drawbacks PRO Very high throughput Di-base ligation ensures built-in accuracy check Low error rate for low-coverage Can handle repetitive regions easily CON Strong cycle-dependent biases (can be modeled and partly overcome – see Wu et al, Nature Methods, 2011) Low quality color calls (Phred < 20) are common Reported problems with paired ends – most mapped tags don’t map to the same chromosome
33. Ion Torrent Sample Prep Emulsion PCR loads copies of unique sequences onto beads One bead is deposited in each well of a micro-machined plate
34. An Ion Torrent Chip From Ion Torrent promotional material
35. When a nucleotide is incorporated into a strand of DNA by a polymerase, a hydrogen ion is released From Ion Torrent promotional material
36. Ion Torrent Sequencing Process From Ion Torrent promotional material As in 454, nucleotides are washed over the nascent strand in a prescribed sequence. Each time a nucleotide is incorporated, hydrogen ions are released and detected.
37. Newest Machine – Ion Proton $150K per machine Ion Proton I chip has 165 million sensors Intended for exomes Ion Proton II chip has 660 million sensors 50X more than 318 chip Claim $1K genome this year
38. Ion Torrent Signals Like 454, a series of pH signals over time as different nucleotides are added From promotional literature
39. Ion Torrent Signals Like 454, the reads don’t always make integer multiples, and some guessing is needed
40. Ion Torrent Advantages & Drawbacks Homopolymer error rates PRO Very high throughput potential Very fast (an afternoon) CON Homopolymerrun errors are still a problem, but less so recently Very uneven loading of sequences wastes a lot of real estate on the chips No prospect of paired-end reads Loading Density
41. Newer Technologies Complete Genomics Pacific Biosciences Oxford Nanopore
42. Complete Genomics Service company only – no equipment sales ~$4,000 per human genome (2011 price) DNA Nanoball technology generates paired-end sequences plated at high density Sequenced by ligation
43. Pacific Biosciences Single-molecule real-time (SMRT) sequencing by circular strand technology using semiconductor technology Long reads promised at under $200 per genome High random error rates reported early Seems better now
44. Signals from Pac Bio Can Detect mC From Agarwal et al, Nature Methods
45. Oxford Nanopore Single-molecule sequencing by threading DNA through a protein nanopore GridION is a general technology for sequencing polymers by measuring current – can do polypeptides also