2016. Whole Genome Sequencing

10/2/2016 Objectives The How and Why of Next Generation Sequencing in Personalized Medicine Pat Tille Ph.D. MLS(ASCP) FACSc • Describe the overall a...
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10/2/2016

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 Evolution of Sequencing • • • • • •

Maxim-Gilbert Sequencing Sanger Dideoxy Sequencing Slab-gel electrophoresis Capillary electrophoresis Shot gun sequencing High-through put sequencing

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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

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Medical and Diagnostic Applications •

Genome Sequencing • • • • • • •

Denovo sequencing Resequencing Targeted resequencing Mitochondrial sequencing Mutation detection Amplicon sequencing Amplicon panels

• Transcript Expression Profiling • •

RNA sequencing miRNA sequencing

• Transcription Factor Binding • Structural Variations • Metagenomics

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

• • • •

Molecular Oncology Infectious Disease Inherited Diseases Pharmacogenetics

Laboratory Medicine is quickly evolving into the era of METAGENOMIC MEDICINE!

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