Targeted Sequencing in the NBS Laboratory Christopher Greene, PhD Newborn Screening and Molecular Biology Branch Division of Laboratory Sciences Gene Sequencing in Public Health Newborn Screening February 16, 2017 National Center for Environmental Health Division 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 Overwhelming There are many things to consider How will results be used Assay Cost Bioinformatics and Analysis Design for Molecular Space Gene Panel Single Gene Birth Rate What is the best sequencing approach for my program? Turn Around Time Prevalence Other Options What to Report Staff Expertise Regulatory Requirements Equipment
General DNA Sequencing Workflow All sequencing approaches have a similar workflow Laboratory Sample Prep/ Extraction Analysis/Informatics Data Filtering and Assembly Library Preparation Variant Calling Sequence Generation Interpretation and Reporting Each of these influence the decision of which instrument to use
Newborn Screening for Select Conditions that use Sanger Sequencing Technology Disorder # of PCR Amplicons # of Sequencing Reactions Total Length Sequence (bp) Pompe 14 34 8600 Krabbe 18 43 9900 MPS1 15 30 5500 X-ALD 7 46 5500 CFTR 42 86 18000 HBB 1 10 1900 HBA1 1 10 1600 HBA2 1 10 1600 VLCAD 12 40 5300
CFTR Sanger Sequencing Workflow 42 PCR reactions/sample Setup 1.5 hours, PCR run time 2.5 hours 86 Cycle Sequencing reactions/sample Setup 45 min, Run time 2.5 hours Manual Electropherogram Analysis: 1+ hour pending on complexity of sequence 48 channel Capillary sequencer Injection time 45 min
Capillary Sanger Sequencing Advantages 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 Sequencing Advantages 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
Ion Torrent Technology Direct conversion of released H + into digital information 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 Slide complements of Life Technologies
Ion Torrent Method Highlights 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
TrueSeq Technology Adapted from Lu et al, Next Generation Sequencing Advances, Applications and Challenges, 2016
TrueSeq Method Highlights 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
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 18 Ion PGM - 316 1 GB 180 Ion PGM - 318 2 GB 360 Ion S5-520 2 GB 360 Ion S5-540 8 GB 1440 Ion S5-560 15 GB 2700 Illumina MiniSeq 8 GB 1440 Illumina MiSeq 15 GB 2700 Illumina 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 Instrument Costs* Instrument List Price Reagents per 8 Samples Chip/Array plus Reagent Cost Ion PGM + Ion Chef $50,000 ~$480 - $800 $350 - $750 Ion S5 + Ion Chef $125,000 ~$480 - $800 $930 - $1250 Illumina MiniSeq $49,500 ~$800 $550 - $1500 Illumina MiSeq $99,000 ~$800 $230 - $1500 Illumina MiSeqDx $125,000 ~$800 $545 - $3600 ABI 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 Overwhelming There are many things to consider How will results be used Assay Cost Bioinformatics and Analysis Design for Molecular Space Gene Panel Single Gene Birth Rate What is the best sequencing approach for my program? Turn Around Time Prevalence Other Options What to Report Staff Expertise Regulatory Requirements Equipment
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 Sufficient library quantity and proper size range Minimum coverage of targeted regions Bioinformatics QC Sample Preparation Library Preparation Sequence Generation Sequence Analysis Result Reporting Sufficient DNA quality and quantity Average read quality scores and metrics Updated mutation databases and interpretative guides Adapted from Gargis, Quality Assurance and Validation of Next-Generation Sequencing
Clinical Sequencing Guidance Published 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 FDA CAP Molecular Pathology Checklist 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 Resources for 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
Thank you! Newborn Screening Saving Lives. Promoting Healthier Babies. Protecting our Future. For more information please contact Centers for Disease Control and Prevention 1600 Clifton Road NE, Atlanta, GA 30333 Telephone, 1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348 E-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. National Center for Environmental Health Division of Laboratory Sciences