Human genome sequence

Transcription

1 NGS: the basics

2 Human genome sequence June 26th 2000: official announcement of the completion of the draft of the human genome sequence (truly finished in 2004) Francis Collins Craig Venter HGP: 3 billion $ 15 years Costs: Celera: 200 million $ 2 years

3 2004: 2 Requests for Application NIH Current technologies are able to produce the sequence of a mammalian-sized genome of the desired data quality for $10 to $50 million; the goal of this initiative is to reduce costs by at least two orders of magnitude. It is anticipated that emerging technologies are sufficiently advanced that, with additional investment, it may be possible to achieve proof of principle or even early stage commercialization for genome-scale sequencing within five years. A parallel RFA solicits grant applications to develop technologies to meet the longer-term goal of achieving four-orders of magnitude cost reduction in about ten years, so that a mammalian-sized genome could be sequenced for approximately $1000.

4 Increased efficiency: decreased costs Exponential cost decrease

5 Efficient integration of each individual step to slash down the costs

6 Massively parallel sequencing Next generation sequencing Key: direct sequencing of DNA without the bacterial cloning step From colonies to polonies

7 454 Roche GS Flex

8 454: Library preparation

9 Clonal amplification of single molecules Emulsion PCR

10 454: Sequencing by pyrosequencing

11 GS Flex throughput ( ) Up to a million sequences 700 bp long (up to 1 kb) in 23 hours

12 454: Game over! Jonathan Rothberg: In the sequencing business, one needs to innovate or die. At 454 we were always first; first non-bacterial cloning, first commercialization, first next-gen individual human genome, Neanderthal, mammoth, deep sequencing, cancer sequencing, drug response studies, HIV, metagenomics, first drug target by whole genome sequencing, and many more firsts. Always innovating, always first."

13 454: Game over! In 2007, Roche acquired 454 for $155 million in cash and stock. Rothberg said that when Roche bought 454, the company was "two years ahead of everyone else," but after the purchase, "they lost that lead, no more firsts, no more innovation."

14 Rothberg strikes back! Rothberg: "When I woke up and found Roche had bought 454 without me, I had to restart. It cost three years. We had to invent a new scalable way to sequence ion semiconductor sequencing and establish a clear path towards both truly low-cost and mobile sequencing." He went on to found Ion Torrent, which was bought by Life Technologies in 2010 for $375 million in cash and stock, and another $350 million based on milestones.

15 Ion Torrent

16 Simple Natural Chemistry

17 Fast Direct Detection dntp H + ph Q Sensing Layer Sensor Plate V Bulk Drain Source Silicon Substrate To column receiver Rothberg J.M. et al Nature doi: /nature10242 Nucleotides flow sequentially over Ion semiconductor chip Direct detection of natural DNA extension A few seconds per incorporation

18 Scalable Semiconductor Technology Wafer Semiconductor Manufacturing Chip Semiconductor Packaging Chip Cross Section Semiconductor Design

19 The Chip is the Machine Scalability Simplicity Speed

20 Two machines, 5 chips PGM Proton P1 P2?

21 Ion Torrent Specs 314 Chip: 0.4 to 0.5 million reads, 30 to 100 Mb data 316 Chip: 2 to 3 million reads, 300 to 1000 Mb data 318 Chip: 4 to 5.5 million reads, 0.6 to 2 Gb data 200bp or 400bp reads, 2 to 7 hours Proton P1: million reads, up to 10 Gb data 200bp reads, 2-4 hours Proton P2: L arlésienne!

22 Barcode read just before insert with Ion Torrent Barcoded adapter Insert Biotin adapter Sequencing primer Barcode

23 Ion Torrent paired-end sequencing

24 Illumina genome analyzer, HiSeq, Miseq (formerly Solexa)

25 Solexa amplification step Amplification and sequencing on a solid support

26 Sequencing by synthesis CRT: cyclic reversible termination

27 Sequencing by synthesis Amplification and sequencing on a solid support

28 120 tiles per lane 480 images per lane and cycle: 36nt run = 138,240 images = 945 Gb 2x50nt run = 384,000 images = 1.3 Tb 2x100nt run = 768,000 images = 5.3 Tb Illumina: Primary data analysis

29 Image analysis (Illumina) Image registration: Get image coordinates congruent Register images between cycles A C Cluster identification Template of cluster positions created from first five cycles G T

30 Cluster identification If neighboring clusters have identical sequences during first 5 cycles: one cluster If neighboring clusters have different sequences during first 5 cycles: two clusters As a consequence: Barcodes should not be included in the first bases otherwise the probability of fusing two different clusters would be too high

31 Illumina paired-end sequencing

32 Barcoding with a single index (Illumina)

33 Barcoding with dual indexing (Illumina)

34 Illumina-Solexa throughput (End 2013) Up to 3 billion sequences, up to 2*100 bp long in 11days (Hiseq2000) Or 0.6 billion, 2*150 bp, in 40 hours (Hiseq2500) Or million, 2*250, in 39 hours (Miseq V2) Or million, 2*300, in 65 hours (Miseq V3)

35 Solid sequencing Applied Biosystems

36 Solid Applied Library

37 Solid Applied Library Emulsion PCR

38 Solid Applied Library

39 Solid Applied Sequencing

40 Solid Applied Sequencing

41 Solid throughput (Early 2009) Up to 0.2 billion sequences up to 2*60 bp long in 7 days

42 Step1: fragment tagging Complete Genomics A human genome for 5,000$

43 Complete Genomics Step2: Clonal DNA amplification A human genome for 5,000$

44 Complete Genomics Step3:Distribution over patterned substrate 1 billion spots per slide A human genome for 5,000$

45 Complete Genomics Step 4: Sequencing by ligation A human genome for 5,000$

46 Step 5: Assembly Complete Genomics A human genome for 5,000$

47 Complete Genomics Costs slashing: small volumes, «simple» equipment A human genome for 5,000$

48 Third Generation sequencing Single molecule sequencing No PCR amplification

49 Helicos Bioscience Single molecule fluorescent sequencing on a flow cell

50 Helicos Cyclic reversible termination: single DNA molecule extended one base at a time, blocking fluorescent label removed and washed, and reiteration

51 Helicos Improved cyclic reversible termination and single DNA molecule detection

52 Helicos throughput Up to 1 Billion sequences On average 32 bp long in 7 days

53 Pacific Biosciences Long single molecule sequencing

54 Pacific Biosciences The label is on the phosphate, and the label is captured transiently using a DNA polymerase tethered on a nanopore

55 Pacific Biosciences Thousands of nanoguides concentrate light The ZMW nanostructure provides excitation confinement in the zeptoliter (10 21 liter) regime

56 Label on the phosphate, not on the base Pacific Biosciences

57 Pacific Biosciences Real time detection of incorporation of each base on thousands of molecules

58 Pacific Biosciences throughput Each pore: 10 bases/sec Claim: in 2013, a high quality human genome in 15 minutes

59 Third or Fourth generation sequencing Single molecules, no fluorophore Oxford Nanopore Technology

60 Oxford Nanopore Exonuclease Pore across lipid bilayer Nanopore Array chip Bases passing through the pore generate a change in the electrical conductance of the membrane allowing electrical measurements. A, T, G, C and MeC can be distinguished.

61 Oxford Nanopore

62 There are several more possibilities in the pipelines BioNanomatrix VisiGen Dover Systems Intelligent Bio-Systems ZS Genetics Reveo LightSpeed Genomics