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Objectives The How and Why of Next Generation Sequencing in Personalized Medicine Pat Tille Ph.D. MLS(ASCP) FACSc
• Describe the overall a...
Objectives The How and Why of Next Generation Sequencing in Personalized Medicine Pat Tille Ph.D. MLS(ASCP) FACSc
• Describe the overall application of NGS to predictive and personalized medicine • List strengths and weaknesses of NGS
SDSU Program Director ASCLS Region V Director
This is a printable version, that does not contain presentation color graphics.
• Describe the overall difference of NGS in comparison to the traditional implementation of a laboratory test
Whole Genome Sequencing • Whole genome sequencing provides information about coding and noncoding parts of a genome. • To identify important pathways. • For evolutionary studies and species comparison. • For more effective personalized medicine (why a drug works for person X and not for Y). • Disease-susceptibility prediction based on gene sequence variation.
The History of Sequencing • Allan Maxim and Walter Gilbert developed an important method of DNA sequencing in 1976-1977. • This method of chemical modification of DNA was technically complex and used extensive hazardous chemicals. • Difficulties with scale-up.
Shotgun Sequencing
Sanger Sequencing •
Sanger developed the chain-termination method of DNA sequencing in 1977.
•
Method was used for fairly short strands (100 to 1000 base pairs) and longer sequences were subdivided into smaller fragments.
•
The small fragments were then subsequently re-assembled into the overall sequence
Technical Challenges
• Shotgun sequencing was developed for
sequencing of large fragments of DNA in 1979.
Removal of artifacts
• DNA is broken up randomly into
numerous small segments, which are sequenced using the chain termination method producing short reads.
Genome assembly
• Shotgun sequencing led to full genome
sequencing. Annotation and validation of assembled genome
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Next Generation Sequencing • Sequence full genome of an organism
in a few days at a very low cost. • Produce high throughput data in the
form of short reads.
• Bacteriophage fX174, was the first genome to be sequenced, a viral genome with only 5,368 base pairs (bp). sequenced
Ion torrent
Roche’s 454
Illumina
ABI’s SOLiD
Data (Mb per run)
100
100
600
700
Time per run
1.5 Hrs
7 Hrs
9 Days
9 Days
Read length
200 bp
400 bp
150 bp
75 bp
Cost per Mb
5$
84.39 $
0.03 $
0.04 $
Genome Annotation
NGS Implementation
• First bacterial genome Haemophilus influenza.
Technique
was
• The first nearly complete human genomes sequenced were J. Craig Venter's, James Watson's, a Han Chinese, a Yoruban from Nigeria, a female leukemia patient, and SeongJin Kim.
• A process of attaching biological information to sequences (contigs or chromosomes). • Consists of two main steps: • Identifying elements on genome a process called gene prediction (Structural annotation) . • Attaching biological information to these elements (Functional annotation).
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Genome Annotation •
LIBRARY PREPARATION
Structural annotation
ORFs and their localization Gene structure Coding regions Location of regulatory motifs
EMULSION PCR
• Functional annotation • Biochemical function Biological function Involved regulation and interactions Expression
Semi-conductor Sequencing Ion Torent Pyro Sequencing 454
Technical Challenges • • • • •
High Complexity Errors in Amplification Contamination G-C Base Pair Bias Is every gene or target present in library (unampifiable) • Secondary structures-read through • Low quality DNA- extraction or other types of samples (Forensic, archealogical) • Information Processing (supercomputing)
POLONY PCR ON A SLIDE
Sequencing by Ligation SOLiD Reversible Terminator Sequencing Illumina
Technical and Application Advantages • • • • • •
Rapid sequencing Information available Pharmacogenomics Exome Sequencing Transcriptome Sequencing Applications beyond basic knowledge of sequences • Third Generation Sequencing has no GC Bias, no library development, sequence RNA directly
Sequencing to the Laboratory • Can next generation solve the problem or diagnostic questions? • What application is needed? (Denovo versus exome or transcriptome or structural) • What is the best platform? Technical differences? Do I need more than one? • What about data analysis? Bioinformatics? Software? Free or purchase? • Coverage?
Implementation • Coverage? • •
How much of the genome is actually included? Multiple comparisons to result (1X versus 3X)
• Garbage in and Garbage out • • •
Contamination Sample degradation Mixed samples
• Control material •
Biobanks
• Statistical Accuracy
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Quality Initiatives • Next Generation SequencingStandardization of Clinical Testing (NexStoCT) • Genetic Testing Reference Materials Coordination Program (GeT-RM) • • •
Quality Control Proficiency Testing Test Development and Validation
●454 Sequencing / Roche −GS Junior System −GS FLX+ System ●Illumina (Solexa) −HiSeq System −Genome analyzer IIx −MySeq ●Applied Biosystems - Life Technologies −SOLiD 5500 System −SOLiD 5500xl System ●Ion Torrent - Life Technologies −Personal Genome Machine (PGM) −Proton ●Helicos −Helicos Genetic Analysis System ●Pacific Biosciences −PacBio RS ●Oxford Nanopore Technologies −GridION System −MinION