Bio conference live 2013

What to use for targeted re-
sequencing – microarrays or Next
Generation Sequencing?
Agnieszka M. Lichanska
TPCaruso & associates, Durham, NC
Choices
for
gene-c
tes-ng

Content
1.  Learning objectives
2.  What is re-sequencing?
•  Why do we use it?
•  When do we use it?
•  What are the types of re-sequencing methods?
3.  Technologies used for re-sequencing – technical comparison
•  Workflows
•  Comparison of performance
•  Data analysis
4.  Establishment of a new assay in the laboratory
•  Requirements
•  Validation
•  Reference materials
•  Reporting and interpretation
•  Regulatory oversight
5.  Summary
6.  Questions
TPCaruso & associatesBioConference Live Genetics and Genomics 2013

Learning Objectives
1.  Understand how targeted re-sequencing is
applied to genetic screening
2.  Appreciate the complexity of validation protocols
for re-sequencing assays.
BioConference Live Genetics and Genomics 2013 TPCaruso & associates

Re-sequencing
•  It is sequencing of a part or whole genome
of an individual in order to detect
sequence differences between the
individual and the standard genome of the
species
BioConference Live Genetics and Genomics 2013
TPCaruso & associates

Re-sequencing
•  When to use it?
o  Carrier screening
o  Prenatal screening
o  Newborn screening
o  Undiagnosed childhood diseases/disorders
o  Risk assessment for complex diseases
•  Types of methods used:
o  Targeted re-sequencing panels
o  Exome sequencing
o  Whole genome sequencing
•  Applications:
o  Genotyping
o  Variation screening

How to perform targeted re-sequencing?
Exon

Regulatory
region

Amplicon

Flanking
region

Buﬀer
region

Primer

Target

Amplicons

Probe

Gene
X,
area
of
interest

DNA

Beads

Gene
X,
area
of
interest

DNA

Array

Probe

In
solu(on
capture
On
array
capture

PCR
ampliﬁca(on

MPX
PCR
&
LR_PCR

RainDance
ePCR

Fluidigm

Haloplex

Ion
Torrent

Illumina

Changing face of genetic testing
Complexity

Number
of
genes

1

>10,000

10

100

1000

Low
Medium
High

Sanger

Arrays

NGS

Assays available for targeted re-sequencing
Technique
Assays

Sanger
Sequencing
ABI
Resequencing
Sets
(RSSs)

Array-‐based
high-‐throughput
re-‐
sequencing
strategies

Pan-‐Ethnic
Carrier
Screening
Panel

*Noonan
Syndrome

*Cardiac
myopathy

*Charcot-‐Marie
Tooth

Next
GeneraUon
Sequencing
Carrier
screening
panel,
incl.
CFTR
panel

Noonan
syndrome
panel

Cardiac
diseases
panel

Pharmacogenomics
panel

Intellectual
disability

AuUsUc
disease
spectrum
panel

Pulmonary
diseases
panels

NeurodegeneraUve
disorders

MulUple
cancer
panels
and
more

*
The
assay
is
no
longer
used,
transiUoned
to
NGS

TECHNICAL COMPARISON

Re-sequencing microarrays
•  Formats and total number of bases
that can be sequenced
o  Cartridge:
o  49 format – 317 kb
o  Strips of 4 chips
o  96-well plates of chips (High Throughput
Assay or HTA)

TPCaruso & associates
1234!
GAGACGGATTCTCCGCCGAGCTGTCCGATACG!
CTCTGCCTAAGAGGCGGCTCGACAGGCTATGC!
Reference sequence
Patient’s Sample
sequence
GAGACGGATTCTCCACCGAGCTGTCCGATACG!
------------GGTG----------------!
GAGACGGATTCTTCGCCGAGCTGTCCG!
GAGACGGATTCTGCGCCGAGCTGTCCG!
GAGACGGATTCTCCGCCGAGCTGTCCG!
------------GGTG-----------!
GAGACGGATTCTACGCCGAGCTGTCCG!
AGACGGATTCTCTGCCGAGCTGTCCGA!
AGACGGATTCTCGGCCGAGCTGTCCGA!
AGACGGATTCTCCGCCGAGCTGTCCGA!
-----------GGTG------------!
AGACGGATTCTCAGCCGAGCTGTCCGA!
GACGGATTCTCCTCCGAGCTGTCCGAT!
GACGGATTCTCCGCCGAGCTGTCCGAT!
GACGGATTCTCCCCCGAGCTGTCCGAT!
GACGGATTCTCCACCGAGCTGTCCGAT!
----------GGTG-------------!
ACGGATTCTCCGTCGAGCTGTCCGATA!
ACGGATTCTCCGGCGAGCTGTCCGATA!
ACGGATTCTCCGCCGAGCTGTCCGATA!
---------GGTG--------------!
ACGGATTCTCGCACGAGCTGTCCGATA!
Base # 1
Base # 2
Base # 3
Base # 4

Re-sequencing microarrays difficult sequences
Reference sequence
GAGACGGATTCTCC---GAGCTGTCCGATACG!
------------GG---CTC------------!
GAGACGGATTCTCCGTTTCCGAGCTGTCCGATACG!
------------GGCAAAGGCTC------------!
Reference sequence
GAGACGGATTCTCCTCCTCCGGGGGGTCCGATACG!
CTCTGCCTAAGAGGAGGAGGCCCCCCAGGCTATGC!
GAGACGGATTCTCCTCCTCCGGGGGGGGTCCGATACG!
CTCTGCCTAAGAGGAGGCAGCCCCCCCCAGGCTATGC!
Repeats
Single base changes
in homopolymer
stretches
Insertions
Deletions
Large deletions
Exon
1
Exon
2
Exon
3

Next Generation Sequencing
36
-‐
250bp
Up
to
200bp
Up
to
1000bp
50
–
75bp
Up
to
20,000bp

Fluorescence
H+
ion
detecUon
Luminescence
Fluorescence
Fluorescence

Bridge

ampliﬁcaUon

ePCR
ePCR
ePCR
No

ampliﬁcaUon

Homopolymer

errors

Homopolymer

errors

slow
Low
yield
at

high
accuracy

Signal/Noise

RaUo

Sequence

Sequencing workflows – Microarray vs NGS
DNA
FragmentaUon

Sequencing

Data
Analysis

ReporUng

gDNA
template

MPX
PCR
amplificaUon

Pooling,
purificaUon,

FragmentaUon,
labeling

HybridizaUon

Wash
&
stain

Scanning

Targeted
enrichment:*

Capture
or
amplificaUon

Tagging
of
the
fragments*

*
Order
of
steps
varies
on
a

protocol
used

Microarray NGS

NGS output example
CTTACAGATATGTGTTGAGACGGATTCTCCGCCGAGCTGTCCGATACGCCCATGAAAAGCT!
AARS
c.986G>A

CTTACAGATATCTGTTCA

CTTACAGATATCTGTTGAGA

CTTACAGATATCTGTTGAGACGGAT

CTTACAGATATCTGTTGAGACGGAT

CTTACAGAAATCTGTTGAGACGGAT

CTTACAGATATCTGTTGAGACGGATTCT

CTTACAGATATCTGTTGAGACGGATTCTCC

CAGATATCTGTTGAGACGGATTCTCCACCG

CAGATATCTGTTGAGACGGATTCTCCACCG

GATATCTGTTGAGACGGATTCTCCACCGAG

ATAT-TGTTGAGACGGATTCTCCACCGAGC

GATATCTGTTGAGACGGATTCTCCACCGAG

TGTTGAGACGGATTCTCCACCGAGCTGTCC

TGTTGAGACGGATTCTCCACCGAGCTGTCC

GAGACGGATTCTCCACCGAGCTGTCCGATA



GATTCTCCACCGAGCTGTCCGATACGCCC

GATTCTCCACCGAGCTGTCCGATAGGCCC

TCTCCACCGAGCTGTCCGATACGCCCATG

CTCCACCGAGCTGTCCGATACGCCCATGA

CTCCACCGAGCTGTCCGATACGCCCATGA

TCCACCGAGCTGTCCGATACGCCCATGAA

TCCACCGAGCTGTGCGATACGCCCATGAA

REFERENCE
SEQUENCE

Coverage

COMPARISON OF MICROARRAY-BASED
ASSAYS AND NGS-BASED ASSAYS
Pros and cons of each technology

Pros and cons of re-sequencing
microarrays
Pros
•  Quick turn around time –
24-48 hrs
•  Simple experimental
protocol
•  Once optimized rapid
bioinformatic analysis
•  Reports sequence at each
tested locus unlike SNP
arrays
Cons
•  Insertions and deletions are a
problem
•  Requires MPX PCR
amplification for targets
•  Noise is not consistent across
the array
•  Costly modifications to
content
•  Mutations can only be
detected in tiled regions
•  Problematic templates:
–  Partly degraded DNA
–  Whole genome amplified DNA

Pros and cons of NGS
Pros
•  Detection of insertions
and deletions
•  Detection of novel
mutations
•  Better reproducibility
•  Less incidental findings
•  Continually decreasing
pricing
Cons
•  Not full coverage in
some areas
•  More complex sample
preparation
•  Development of
bioinformatic analysis is
complex
•  Sequence length can
be an issue
•  Risk of over-
interpretation of results

SUMMARY COMPARISON OF MICROARRAY-
BASED ASSAYS AND NGS-BASED ASSAYS
Feature Microarray NGS
Development time Same as NGS – 50% Shorter – 50%
Turn around time In majority cases shorter
Reproducibility - 100%
Data Analysis
Data complexity
Pipeline/report development
More accidental findings
More pathogenic mutations
Higher confidence in data
No difference – 50%
Similar
50%
Similar
No
Yes
Yes
Data Quality:
Less “N” calls
Substitutions detection
Indels detection
-
Same
Not adequate
100%
Same or slightly better
Better
Cost effectiveness Varies Varies
Validation Similar or slightly slower Similar or slightly quicker

Data Analysis/Reporting
Sequence
1.  QC
•  Experiment
•  Obtained sequences
•  Remove poor sequences
2.  Align to the reference genome
3.  Filter out known SNPs (non-
pathogenic)
4.  Identify mutations
5.  Create a report
•  Sample name
•  Test name
•  Laboratory QC metric
•  List of mutations, zygosity
•  Frequency of alleles
Sample Report
data view: 16bp deletion
bp deletion visualized by aligning
e p53 reference sequence.
bp deletion visualized by aligning
he p53 reference sequence.
Fig. 7 . Heterozygous T>G mutation
visualized by SeqNext by looking at
the actual reads
Fig.8. SeqNext can print a clinically
relevant report

WHICH ONE TO CHOOSE THEN?
Choice is based on a number of factors:
1.  Required throughput
2.  Nature of mutations
3.  Complexity of the assay to be performed
4.  Availability of equipment and expertise
5.  Time required for implementation of the assay

ESTABLISHING TARGETED RE-
SEQUENCING ASSAY IN THE
LABORATORY
1.  Reasons for introduction
2.  Choice of platform
3.  In-house development or off the shelf product
4.  Validation – technical and clinical
5.  Implementation

1. Reasons for introducing a new assay
•  Streamline the tests available – instead of 10s of tests
just one
•  Provide more comprehensive testing
•  Meet the clinical needs of the customers
•  Better performance, more accurate, more specific
•  Competition already using gene panels
•  Improve turn around time
FASTER – BETTER - CHEAPER

Optimization of
a test
VALIDATION
ASSAY
PERFORMANCE
QC
PROFICIENCY
TESTING
DEVELOPMENT
Platform
Assay/Test
IT/pipeline
Patient testing
IMPLEMENTATION
Daily
or
when test is run
Periodical•  Assay
content

•  OpUmizaUon
of
capture

or
ampliﬁcaUon

•  OpUmizaUon
of

sequencing
process

•  Technical
validaUon

•  Development
of
analysis

Platform and assay source
2. Choice of platform
NGS vs microarrays
Type of NGS instrument
Enrichment method
3. In-house development, off the shelf product or collaboration with
another company
Regulatory requirements
Time and costs of assay development
Time and costs of assay validation
Availability of assay analytical performance documentation

4. Validation of a re-sequencing assay
•  Definition of performance specifications
–  Analytical sensitivity – likelihood of detecting
sequence variation if present
–  Analytical specificity – false +ve rate
–  Reportable range – genes, exons, genomic regions
–  Reference range – reportable sequence variations
that can be detected
–  Necessary coverage – to make accurate calls
•  Establishment of QC process for the performance
specs
–  Coverage, quality scores, strand bias, GC bias,
transition/transversion ratio, allelic read percentage
•  Establishment of proficiency testing (PT)

Reference materials – critical part of
validation and on-going QC protocols
•  gDNA (blood or cell lines)
–  Blood (non-renewable)
–  Cell lines (renewable) but can have genetic
aberrations
–  Similar to patient’s sample
•  Synthetic
–  Not representative of gDNA complexity
•  Electronic reference materials
–  Reference for analysis steps
–  Data files might not be exchangeable between
platforms

Example of reference materials

5. Implementation
•  Regulatory oversight
–  FDA requirements – LDT vs cleared/approved tests
–  CLIA regulations
–  State requirements
•  Guidance
–  CDC
–  Clinical Lab Standards Institute
–  ACMG
–  AMP
–  CAP
•  Proficiency testing and QC
–  No formal programs exist for NGS
–  QC has to include sample prep, library prep, generation of
sequences, analysis of sequences and result reporting

Implementation – Reporting guidance
•  Constitutional mutations in genes (57) listed
by ACMG on a minimum list – reported
independently of the test indications
•  Additional genes – might be analyzed for
incidental findings (up to the lab)
•  Incidental findings – report irrespective of the
patient’s age
•  Incidental findings – reported for a normal not
tumor tissues

Who is going to provide counseling to
the patient?
•  According to ACMG the ordering clinician or
their team is responsible for providing the
counseling
•  A few points on this:
–  Clinicians need to be familiar with NGS and its
limitations
–  Explain to the patients a possibility of incidental
findings
–  A clinical geneticist should be consulted regarding
the ordering, interpretation and communication of
the complex NGS assays.

Summary
•  Targeted sequencing allows sequencing of
panels of genes for particular conditions
•  Development and validation are complex
–  Reference genome
–  Reference templates
–  Alternative tests for sequences with no or limited
coverage
•  Reporting requirements
–  Combines QC of the assay and genetic data
–  Simple yet comprehensive

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