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Human Genome Project
Human Genome Project
$3 Billion; >10 years; collaborative effort
Overcoming the limitations
1. Limited room for further cost reductions in conventional DNA sequencing (“Sanger”, Human Genome Project)
Competitive space for development of new ideas and technology
2. Whole genome assemblies available for Human (Human Genome Project) and other major model organisms.
Act as reference genomes against which to map short read sequences
3. Methods developed for a broad range of research topicsGenetic variation, chromosomal rearrangement, epigenetic modification, protein-DNA interaction, RNA expression
4. Technological developmentMicroscopy, chemistry, enzyme engineering, computation, data storage…
5. Demand for future developmentContinued demand for further scaling of output and rapid turnaroundDemand for increasing read lengths, quality and direct/single molecule sequencing
Adapted from Shendure & Hanlee (2008) Nature Biotechnology
Ion sensor detects each base addition due to release of single H+ ion
Non-matching nucleotides do not incorporate AND do not give rise to signal
Homopolymers detected as increased signal due to increased release of H+ ions
The need for label-free, single molecule sequencing
Exonuclease sequencing: Oxford Nanopore's first generation of sequencing technology
Protein engineering – alpha hemolysin detection of DNA bases
Cyclodextrin molecule attached to the inside surface acts as a binding site for DNA bases
Future generations of Oxford Nanopore's sequencing technology:
• The ability to sequence intact strands of DNA rather than cleaving individual bases
• a combination of protein nanopore and man-made membrane (instead of lipid bilayers)
• nanopores as holes made directly in man made materials ("solid state" nanopores)
Future generations of Oxford Nanopore's sequencing technology:
• The ability to sequence intact strands of DNA rather than cleaving individual bases
• a combination of protein nanopore and man-made membrane (instead of lipid bilayers)
• nanopores as holes made directly in man made materials ("solid state" nanopores)
Detect current change as bases enter pore
Electronic readout as bases drop/pass through pore
Real time read out
How do high concentration reagents effect the stability of the pore & bilayer?
Zero Mode Waveguide
Each ZMW is a cylindrical hole, tens of nanometers in diameter, perforating a thin metal film supported by a transparent substrate
ZMW with DNA polymerase
Single DNA polymerase molecule attached to the bottom of the ZMW using a proprietary biased immobilization process
background interference
High concentration of labeled nucleotides creates a fluorescent background thousands of times brighter than the signal of a single incorporation event
SMRT Cell
Each SMRT Cell contains thousands of Zero Mode Waveguides (ZMW)
Moderated Detection Volume
Attenuated light from the excitation beam penetrates 20-30 nm of each waveguide, creating detection volume of 20 zeptoliters (10-21 liters)
Circular consensus sequencing. A strand displacing enzyme on a circular template generates independent reads. The quality score increases linearly with the number of times the molecule is sequenced.
Standard SequencingSingle pass long reads
Circular consensus sequencing. Continued generation of reads based on insert size
Strobed ReadsLasers alternate between on/off generating multiple subreads and extnding run length
Archon Genomics X PRIZE
The era of personalized medicine is dawning
As scientists gain knowledge from mapping the Human Genome, they will also find new ways to treat and even prevent disease. To build the library of information necessary to advance the field of genomic medicine, it is imperative that we develop DNA sequencing technology that is faster and affordable.
Personal Interest:Sequencing “healthy” individuals
Craig Ventor
James Watson
Personal Interest:Sequencing “healthy” individuals
Medical Interest:Disease associations
James Lupski
anonymous
Medical Interest:Disease associations
Variants Identified by exome sequencing:
>95% of all variants investigated here were previously reported SNPs and cannot explain a genetically dominant disease.
Focus group
Non-invasive prenatal diagnostics:
Detecting fetal DNA in circulating maternal plasma
Non-Invasive prenatal diagnostics:
Detecting fetal DNA in circulating maternal plasma
QCMG - Pancreatic Cancer Study
Presentation
Diagnosis
Treatment Plan
Surgery
Adjuvant Therapy
Recurrence
Death
Recruitment Patient ConsentSample Collection (Blood)Recording of Serum MarkersRecording of Pathological Data
Sample Collection (Blood)Recording of Serum MarkersRecording of Pathological Data
Date and Cause
Sample Collection (Blood)Recording of Serum MarkersRecording of Pathological Data
Sample CollectionOperative Data RecordingXenograft Generation
Resection
Prospective study of pancreatic ductaladenocarcinoma. The primary treatment mode for pancreatic cancer is surgical resection. Our major study focuses on the sequencing of pancreatic tumour samples with detailed clinicopathology and outcome data. As well as tumor material, the study includes collection of matched normal tissue, and the generation of xenografts and cell lines for ongoing propagation of tumour material. Early investigation will look to verify the efficacy of xenograft explants and cultured cell lines as representative of the source tumor.
QCMG - Ovarian Cancer Study
Presentation
Diagnosis
Treatment Plan
Surgery
Adjuvant Therapy
Resistance
Death
Recruitment Patient ConsentSample Collection (Blood)Recording of Serum MarkersRecording of Pathological Data
Date and Cause
Sensitivity versus Resistance
Ascites Sample CollectionOperative Data Recording
Remission
Longitudinal study of chemotherapy resistance in serous invasive ovarian cancer.Chemotherapeutic treatment of ovarian cancer (eg. Platinum) is limited by the acquisition of resistance in recurrent tumours. This study includes a longitudinal collection of tumour material from three distinct cohorts. 1. Sensitive primary and relapse tumours; 2. Sensitive primary tumours with acquired resistance on relapse; 3. Resistant primary tumours.
Every cancer is different; every patient is different
Personalized tumor biomarkers for cancer surveillance
Detecting circulating tumour DNA
Personalized tumor biomarkers for cancer surveillance
Detecting circulating tumour DNA
Personalized Medicine
The sequencing of whole human genomes, transcriptomes and methylomes of individual patient samples is a reality
Cancer genome analyses have the potential to identify personalized biomarkers for cancer surveillance and assess the status of key druggable pathways.
Wide array of medical applications for sequencing technologies.
In the past the challenge was to generate sufficient information. The new challenge is to manage, interpret, test and apply it appropriately.
There is huge opportunity in improving analytical, computational & genomic aspects of this research!
“ The Promise of Personalized Medicine
Imagine the day when you and your doctor sit down to review a copy of your own personal genome. This vital information about your biology will enable your physician to inform you of your disease susceptibilities, the best ways to keep yourself healthy and how to avoid or lessen the impact of future illness.
In the past, the diagnostic classification of a cancer was based on the organ or tissue location, such as liver or breast cancer. But now, the many forms of cancer can be characterized by their molecular profile. These molecular characteristics provide new information on how rapidly the cancer might spread or how it might respond to specific treatments.
The use of personal genetic information to predict disease susceptibility and guide proactive
care has the power to transform our entire healthcare system. … ”
