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: cdcinfo@cdc.gov 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

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