-
Targeted Sequencing in the NBS Laboratory
Christopher Greene, PhD
Gene Sequencing in Public Health Newborn ScreeningFebruary 16,
2017
National Center for Environmental HealthDivision of Laboratory
Sciences
Newborn Screening and Molecular Biology BranchDivision of
Laboratory Sciences
-
Disclaimer
The findings and conclusions in this presentation are those of
the speaker and do not necessarily represent the views of the U.S.
Centers for Disease Control and Prevention
Use of trade names is for identification only and does not imply
endorsement by the Centers for Disease Control and Prevention, the
Agency for Toxic Substances and Disease Registry, or the U.S.
Department of Health and Human Services
-
Decision Points can be OverwhelmingThere are many things to
consider
Design for Molecular
Space
Staff Expertise
Birth Rate
Gene Panel
Regulatory Requirements
Bioinformatics and
Analysis
Single Gene
Assay Cost
Prevalence
What is the best sequencing approach
for my program?
What to Report
Turn Around Time
How will results be
used
Other Options
Equipment
-
General DNA Sequencing Workflow
All sequencing approaches have a similar workflow
Laboratory Analysis/Informatics
Each of these influence the decision of which instrument to
use
Sample Prep/ Extraction
Library Preparation
Sequence Generation
Data Filtering and Assembly
Variant Calling
Interpretation and Reporting
-
Newborn Screening for Select Conditions that use Sanger
Sequencing Technology
Disorder # of PCR Amplicons
# of SequencingReactions
Total LengthSequence (bp)
Pompe 14 34 8600Krabbe 18 43 9900MPS1 15 30 5500X-ALD 7 46
5500CFTR 42 86 18000HBB 1 10 1900HBA1 1 10 1600HBA2 1 10 1600VLCAD
12 40 5300
-
CFTR Sanger Sequencing Workflow
42 PCR reactions/sampleSetup – 1.5 hours,
PCR run time 2.5 hours
86 Cycle Sequencingreactions/sample
Setup 45 min, Run time 2.5 hours
48 channel Capillary sequencer Injection time – 45 min
Manual Electropherogram Analysis:1+ hour pending on complexity
of sequence
-
Capillary Sanger SequencingAdvantages and Disadvantages
96-well and 384-well formats for flexible workflow
Reagent use can be optimized per assay
Data analysis does not require sophisticated bioinformatics
“Gold Standard” for DNA sequencing, often used for NGS
confirmation
Sequence repeats and insertions/deletions complicate
analysis
DNA template requirements of 5 - 10ng per amplicon
-
Targeted Next Generation Sequencing
How does it differ from Sanger/traditional sequencing
Massively parallel reaction of up to millions of targets in a
single assay
• Gene panels vs. single gene Samples from multiple
individuals can be multiplexed in a single run
Analysis at level of individual DNA fragments
-
Next Generation SequencingAdvantages and Disadvantages
Reduced complexity of sample and library prep
10ng of DNA per library – single punch for multiple genes
Sequence analysis of insertion and deletion mutations
simplified
Small instrument foot-print
Time to analysis may be longer than Sanger sequencing
Fixed cost per run can lead to wasted capacity
Illumina CFTR assay is FDA approved
Fixed 139-variant panel, requires MiSeqDX instrument
-
A H+ ion is released as each nucleotide is incorporated into the
DNA strand causing a transient pH change
Ion Torrent semiconductor sequencing chip is able to detect this
change in pH and convert it directly to base calls
Ion Torrent TechnologyDirect conversion of released H+ into
digital information
Slide complements of Life Technologies
-
Different chip capacities for scalable throughput
Multiplex up to 384 samples per run
Automated data analysis for selected variants
Simple reagent requirements for lower per sample cost
Up to 400bp of sequence per read
Short reaction time
Ion Torrent Method Highlights
-
TrueSeq Technology
Adapted from Lu et al, “Next Generation Sequencing – Advances,
Applications and Challenges, 2016
-
TrueSeq probe ligation for library construction has
higher specificity than traditional PCR
Single-base extension chemistry suited for difficult
sequence regions
Read-length up to 150bp for MiniSeq, 250bp for MiSeq
Multiplex of 96 to 384 samples – kit dependent
Instruments widely available in many public health
laboratories
TrueSeq Method Highlights
-
Assumptions for Targeted NGS Platform Comparisons
“Average Gene” = 10,000bp How many samples for a single gene in
one run How many “genes” for each sample in one run
500X Coverage Unequal amplification can occur for certain DNA
sequences Ensure that all library fragments sufficiently
represented
Library and run conditions Ion Systems with Ion Chef Library
Prep Illumina Systems using Standard Flow Cell
-
How Many 10kb Genes Per Sequencing Run
Instrument Maximum Sequence per Run
“Average Genes” at 500X*
Ion PGM - 314 100 MB 18Ion PGM - 316 1 GB 180Ion PGM - 318 2 GB
360Ion S5 - 520 2 GB 360Ion S5 - 540 8 GB 1440Ion S5 - 560 15 GB
2700Illumina MiniSeq 8 GB 1440Illumina MiSeq 15 GB 2700Illumina
MiSeqDx 15 GB 2700
*(Run capacity)/(10kb x 500); assume 90% loading efficiency
-
Targeted NGS Assay Time Lines
Sample Preparation 6 to 8 hours for TrueSeq library preparation
12 – 17 hours for Ion Chef preparation, depending on chip size 1 to
2 hours bench-time: number of samples and multiplexing
Sequencer Runs Ion systems: 4 to 6 hours per run plus data
processing Illumina systems: 4-27 hours per run, depending on
amplicon size,
including data processing
Note: The FDA-approved CFTR assay is ~27 hour run time
-
Sequencing Platform and Run Costs*
Instrument Instrument “List” Price
Reagents per 8 Samples
Chip/Array plusReagent Cost
Ion PGM + Ion Chef $50,000 ~$480 - $800 $350 - $750Ion S5 + Ion
Chef $125,000 ~$480 - $800 $930 - $1250Illumina MiniSeq $49,500
~$800 $550 - $1500Illumina MiSeq $99,000 ~$800 $230 - $1500Illumina
MiSeqDx $125,000 ~$800 $545 - $3600ABI 3730xl (Sanger) $250,000 n/a
$525**ABI 3500xl (Sanger) $195,000 n/a n.d.
*Pricing information from: CAP Today, December 2016
** Based on CDC experience
-
Decision Points can be OverwhelmingThere are many things to
consider
Design for Molecular
Space
Staff Expertise
Birth Rate
Gene Panel
Regulatory Requirements
Bioinformatics and
Analysis
Single Gene
Assay Cost
Prevalence
What is the best sequencing approach
for my program?
What to Report
Turn Around Time
How will results be
used
Other Options
Equipment
Staff Expertise
-
Next Generation Sequencing Validation
CLIA Regulations require the establishment of performance
specifications to ensure the analytical validity of test results
prior to patient testing
However, Next Generation Sequencing technology and assay
complexity do not neatly fit into existing categories like other
laboratory tests
Programs will need to identify and define test quality metrics
and develop validation plans
-
Sequencing Quality Control
Adapted from Gargis, Quality Assurance and Validation of
Next-Generation Sequencing
SamplePreparation
Library Preparation
Sequence Generation
Sequence Analysis
Result Reporting
Sufficient DNA qualityand quantity
Sufficient library quantityand proper size range
Average read qualityscores and metrics
Minimum coverage oftargeted regions
Bioinformatics QC
Updated mutationdatabases and interpretative guides
-
Clinical Sequencing GuidancePublished Good Laboratory
Practices
CLSI MM09 – Nucleic Acid Sequencing Methods in Diagnostic
Laboratories
Next Generation Sequencing-Standardization of Clinical Testing
Workgroups• Nat. Biotechnol (2012) 30:1033-1036• Nat. Biotechnol
(2015) 33:689-693
Regulatory Checklists CAP Molecular Pathology Checklist
FDA Use of Standards in FDA Regulatory Oversight of Next
Generation Sequencing
Based in Vitro Diagnostics Used for Diagnosing Germline Diseases
(July 2016)
Use of Public Human Genetic Databases to Support Clinical
Utility for Next-Generation Sequencing Based In Vitro Diagnostics
(July 2016)
-
Next Generation Sequencing Resourcesfor Quality Control
NIST Genome In a Bottle: A highly characterized human genomic
DNA for test development, validation, and QC
RM 8392 – Mother, Father, and Son Trio of Eastern European
Ashkenazim Jewish Ancestry
RM 8391 – Adult son of RM 8392
RM 8398 – Daughter of Utah CEPH family, NA12878
RM 8393 – Son of Chinese Ancestry
-
For more information please contact Centers for Disease Control
and Prevention1600 Clifton Road NE, Atlanta, GA 30333Telephone,
1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348E-mail:
[email protected] Web: www.cdc.gov
The findings and conclusions in this report are those of the
authors and do not necessarily represent the official position of
the Centers for Disease Control and Prevention.
Thank you!
National Center for Environmental HealthDivision of Laboratory
Sciences
Newborn ScreeningSaving Lives.
Promoting Healthier Babies.
Protecting our Future.
Targeted Sequencing in the NBS LaboratoryDisclaimer�Decision
Points can be Overwhelming�There are many things to considerGeneral
DNA Sequencing WorkflowNewborn Screening for Select Conditions that
use Sanger Sequencing Technology�Slide Number 6Capillary Sanger
Sequencing�Advantages and DisadvantagesTargeted Next Generation
SequencingNext Generation Sequencing�Advantages and
DisadvantagesIon Torrent Technology�Direct conversion of released
H+ into digital informationSlide Number 11TrueSeq Technology Slide
Number 13Assumptions for Targeted NGS Platform ComparisonsHow Many
10kb Genes Per �Sequencing Run Targeted NGS Assay Time
LinesSequencing Platform and Run Costs*Decision Points can be
Overwhelming�There are many things to considerNext Generation
Sequencing Validation � Sequencing Quality Control�Clinical
Sequencing Guidance�Next Generation Sequencing Resources�for
Quality ControlSlide Number 23
