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NGS: Coming to a Lab Near You!
An Introduction to
Next Generation Sequencing (NGS)
SNUG 2013
Laurel Estabrooks, PhD, FACMG
VP Genetics Business Development
SCC Soft Computer
What is “DNA Sequencing”?
• DNA sequencing involves
the use of various methods
for determining the order of
the nucleotide bases —
adenine, cytosine, guanine,
and thymine — in a
molecule of DNA.
Exon
Intron
Gene
Exon
DNA Basics
• Bases - In molecular biology and genetics, two nucleotides
on opposite complementary DNA or RNA strands that are
connected via hydrogen bonds are called a base pair.
Adenine (A) forms a base pair with thymine (T) and guanine
(G) forms a base pair with cytosine (C). In RNA, thymine is
replaced by uracil (U), and therefore bonds to Adenine (A).
• Genetic code - the set of rules by which information
encoded in genetic material (DNA or mRNA sequences) is
translated into proteins (amino acid sequences) by living
cells. It is a triplet code in that three nucleotides (a codon)
determine particular amino acids.
Basics of Transcription
and Translation
DNA
mRNA
Protein
Basics of Transcription
and Translation
Intron
Exon
DNA
Transcription and mRNA processing
Intron information is not passed onto processed mRNA
mRNA
Translation
Un-translated region
Protein
Post-Translational Modification
Active Protein
What is
Next Generation Sequencing?
• 1st Generation = Sanger Sequencing
– 2 reads (forward & reverse)
• 2nd Generation = Next Generation Sequencing
– Millions of reads
• 3rd Generation = Single Molecule Sequencing
What is
Next Generation Sequencing?
What is
Next Generation Sequencing?
Major computations performed
with NGS data
• Data assembly with base calling at the level of
individual reads
• Alignment of the assembled sequence to a
reference sequence
• Variant calling
NGS Alignment
Multiple, fragmented sequence reads must be assembled
together on the basis of their overlapping areas.
NGS Technology Terminology
• Read length - the average number of contiguous
nucleotide bases in a polynucleotide sequence
that are produced by a particular sequencing
instrument (14-400)
• Coverage – Number of times a nucleotide base is
read (# followed by X: 300X)
• Call – determination of a given base or base
sequence by a sequencing instrument
• Call Quality – accuracy of the call determination
Base Calling Accuracy
Q Scores
• Base calling accuracy often measured by the Phred Quality Score
(Q score) which assesses the accuracy of a sequencing platform. It
indicates the probability that a given base is called incorrectly by the
sequencer.
• Logorithmic calculation
• Q10
1/10 error rate
• Q20
1/100 error rate
• Q30
1/1000 error rate
Example:
Phred score of 30 (Q30) = probability of an incorrect base call 1 in 1000 times
• Low Q scores can result in an increase in false positive variant calls
There are multiple types of DNA
changes including:
Substitution
Duplication
Inversion
Translocation
Insertion/Deletion (Indel)
SNPs - Single Nucleotide Polymorphisms
– Substitution change in more than 1% of the population
– Considered a common variant
CNVs - Copy Number Variations
– Sections of DNA bases in our genomes that are commonly copied
many times over
– Number of copies may vary from person to person
Applications in Microbiology
• Identifying the species of an isolate
• Defining its properties, such as resistance to
antibiotics and virulence
• Monitoring the emergence and spread of
bacterial pathogens
Phylogenic Map
NGS & Microbiology
Case Study
The NHS Rosie Hospital in Cambridge manages around
6,000 baby deliveries each year. All infants in its special
care baby unit are screened for MRSA when admitted,
and for every week while in the unit.
This routine screening picked up MRSA in 12 infants.
The Lancet Infectious Diseases, Volume 13, Issue 2, Pages 130 - 136, February 2013
NGS & Microbiology
Case Study
The following was performed:
• Bacteria was cultured from swabs and plated on selective
media.
• Antimicrobial susceptibility was tested against an array of
antibiotics.
• Sequencing libraries were prepared from each MRSA isolate,
and amplified.
• Whole genome sequencing was performed using the Illumina
MiSeq sequencer.
The Lancet Infectious Diseases, Volume 13, Issue 2, Pages 130 - 136, February 2013
NGS & Microbiology
Case Study
• All affected infants were treated
• Unit was sanitized
The Lancet Infectious Diseases, Volume 13, Issue 2, Pages 130 - 136, February 2013
NGS & Microbiology Case
Study Results
• 14/17 infants had a new sequence type
ST2371
• Only 20 SNPs varied among the 14 ST2371
isolates
• ST22 is common MRSA sequence type in UK
• ST2371 differs from ST22 isolate by an average
of 550 SNPs
NGS & Microbiology
Case Study
• Short hiatus from outbreaks
• Another outbreak
• Tested all SCBU personnel
The Lancet Infectious Diseases, Volume 13, Issue 2, Pages 130 - 136, February 2013
Case Study Analysis
Case Study: NGS Benefit
• Identification of asymptomatic carrier causing
re-infections
• Upon treatment of carrier, no further
outbreaks
The Lancet Infectious Diseases, Volume 13, Issue 2, Pages 130 - 136, February 2013
NGS in Human Genetics
Next Generation Sequencing
Targeted
Panel
Whole
Exome
Whole
Genome
-smaller
-targets
entire
coding
region
-targets
entire
genome
-incidental
findings
-incidental
findings
target
region
-no
incidental
findings
Incidental Findings
• Findings not associated with the original
trigger for the testing
• Currently under debate regarding whether to
report
• Recent guidelines published from American
College of Medical Genetics and Genomics
Next Generation Sequencing
Test Ordering
Test Interpretation
• Unknown diagnosis
• Suspected diagnosis of
disease with mutational
heterogeneity
• Available variant data
• Patient clinical presentation
• Co-segregation of variant
with clinical issue in family
Interpretation Categories
Pathogenic Mutation
A change that has been previously defined
and is known to result in a given disorder,
disease or phenotype.
Interpretation Categories
Probably/Possibly Pathogenic
Not a defined change, but there is additional
evidence based on
– the gene involved,
– the gene position,
– the type of the variation, or
– the family history
that lends greater likelihood that this could
indeed be the origin of the patient’s clinical
presentation/disorder.
How do you determine a variant
is possibly/probably pathogenic?
Use algorithms to assess how variation within a known
gene would theoretically impact gene integrity, gene
translation, or protein formation
Example online tools:
– PolyPhen 2 http://genetics.bwh.harvard.edu/pph2/ (Polymorphic
Phenotyping - predicts loss of function). PolyPhen-2 is a tool which predicts
possible impact of an amino acid substitution on the structure and function of
a human protein using straightforward physical and comparative
considerations.
– SIFT http://sift.jcvi.org/ (Sorting Intolerant From Tolerant, just computes
>4.55 Mb deletions) SIFT predicts whether an amino acid substitution affects
protein function.
Interpretation Categories
Variant of Unknown Significance
• Do not know the significance at this time
• Incidence WGS>WES
Example of Result Tables
Excerpt of Interpretation
Illustrating Interpretation Categories
Questions?