Download Next Generation Sequencing

Next Generation
Sequencing
Next Generation Sequencing
• Often referred to as massively parallel sequencing
•. It is DNA sequencing technology which has revolutionized genomic
research
• Increasing the speed due to parallel analysis technology, and
increase accuracy which decreased manpower and cost.
• An entire human genome can be sequenced within a single day. In
contrast, the previous Sanger sequencing technology required over a
decade to deliver the final draft
Sanger Method
• Produced
in 1977 by Fred Sanger
• Sanger method relies on a type of dNTP (deoxynucleoside triphosphate)
with hydrogen atom rather than a hydroxyl group, called ddNTP (dideoxynucleoside). When these bases bind, they
terminate replication as they cannot bind
other bases.
• How does Sanger Sequencing work?
• ATGCTCAG
Human Genome Project & Capillary
Electrophoresis
• 1990: The Human Genome Project contributed to the 2nd & 3rd generations of
sequencing
• 1987: Capillary Electrophoresis used Sangers chain termination method, but did not
use gels. The method used fluorescently labelled chain terminating ddNTPs added to a
PCR reaction.
• How does Capillary Electrophoresis work?
• Fragments separated according to size, which is measured by charge
• The more negative the charge, the longer the fragment
• As fragments are pulled towards positive electrode of the capillary, they pass a point where a laser beam triggers a flash of light
from the ddNTP that is characteristic of the base type
• Capillary Electrophoresis contributed to the completion of The Human Genome
Project
Library Preparation
The Library is the DNA fragments that represent the
genome
Types of Library’s
•
•
•
•
Whole Genome Sequence
Exome Sequence
RNA Sequence
Methyl-Sequence
Polymerase Chain Reaction
Used to enrich the library by amplifying a single copy of a piece of DNA
creating thousands to millions of copies
• Process
• DNA melting (denature) – split the double helix
• Annealing – primer builds in 3’ end
• DNA synthesis (elongation) – DNA polymerase bind and synthesize target areas
• Types
• Emulsion PCR
• Polony PCR
Polymerase Chain Reaction
• Emulsion
PCR: PCR variation conducted on a bead surface within tiny
water bubbles floating on an oil solution
• Polony PCR: Combined in-vitro paired tag library with ePCR, microscope
and ligation based sequencing
• Sequenced full E. coli genome
• Both are similar to each other, however the denaturation process differs
Types of Next Generation Sequencing
1. 454 Pyrosequencing
• Light emitted when add a NT to nascent DNA
2. Semiconductor Sequence- Ion Torrent
• Based on detection of H+ ions
3. SOLiD – Sequence by Oligonucleotide Ligation and Detection
• Four fluorescent Oligonucleotides compete for ligation to sequencing primer
4. Illumina Sequencing- Reversible Dye-Terminator Technology
• Simultaneously identifies DNA bases whole incorporating them into nucleic
acid chain
Research & NGS
Types of Researcher's that would benefit from doing Next Generation Sequencing
include:
• Pathology
• Replace conventional characterization of pathogens by morphology, staining
properties and metabolic criteria with a genomic definition of pathogens
• Specifically, NGS was used to reveal and trace an outbreak of methicillin-resistant
Staphylococcus aureus (MRSA) on a neonatal intensive care unit in the UK.
•Microbiology
• It allows microbiologists who are studying microbial genomics to discover entirely
new viruses, develop new strategies to control outbreaks, and monitor hostpathogen interactions
• Specifically, example is microbiology and biotechnology researchers using NGS to
provide noninvasive tests for prenatal screening.
Research & NGS
•Oncology
• There are many ongoing several large scale cancer genome projects around the world. A
fully sequenced genome may provide many benefits including a more precise diagnosis and
classification of the disease, and a more accurate prognosis
• Specifically, cancer researchers investigating the mutational landscape of tumor's through
NGS, which is providing new insights into cancer genome evolution
•Molecular Genetics
• These researchers are studying Genetically Modified Organisms (GMO) and applications for
Genetically Modified Plants (GMP)
• Some recent research projects explored the use of NGS as an alternative tool for meeting
the data requirements for the molecular characterization of GMPs in view of their risk
assessment.
Research & NGS
Two specific researchers in Canada interested in NGS products from NuGEN are;
Ontario Institute of Cancer Research (OICR)
• Location: Toronto, Ontario
• OICR is dedicated to research on the prevention, early detection, diagnosis, and treatment
of cancer. OICR has been using NGS into order to develop new medication and target specific
facts about certain cancers.
• Lars Jorgensen, PhD Director of the Genome Sequence Informatics Group OICR, “The
massive amounts of data produced by genome sequencing have allowed scientists to better
understand cancer and how to treat it.”
• Dr. Philip Awadalla, a Professor at University of Toronto, and OICR Senior investigation, builds
on the importance of NGS by sharing his research publication; ‘Variation in Genome-wide
mutation rates within and between human families’.
Research & NGS
BC Genome Sciences Centre
• Location: Vancouver, BC
• They are a leading international center for genomics and bioinformatics research. Their
mandate is to advance knowledge about cancer and other diseases, to improve human
health through disease prevention, diagnosis and therapeutic approaches, and to realize
the social and economic benefits of genomics research.
• An ongoing research project taking place at this research facility is ‘The identification of
mutation specific inhibitors through whole genome re-sequencing of breast cancer celllines’. The leader of the project, Steven Jones, is receiving funding for this project from the
National Cancer Institute of Canada to use nascent next-generation sequencing
technology to derive the complete genomic sequence of breast cancer cell lines.