Generalization of an Assay System using Repeat-primed PCR ...€¦ · SeqStudio Genetic Analyzer*...
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160 120 80 40 0 160 120 80 40 0 0 20 40 60 80 100 120 140 160 180 200 Expected Allele Repeat # SeqStudio Allele Repeat # C9orf72 FMR1 200 180 160 140 120 100 80 60 40 20 0 10000 1000 100 10 CGG 30 CGG 56 CGG 200 Major Minor Repeat Signal Detection vs % Minor Allele in FMR1 Sensitivity Control Signal Intensity (rfu) Repeat # 25% 20% 10% 5% 1% CGG 200 180 160 140 120 100 80 60 40 20 0 10000 1000 100 10 CGG 18 CGG 30 CGG 32 CGG 56 CGG 85 CGG 116 CGG 200 Repeat Signal Detection vs. Input: FMR1 PC Signal Intensity (rfu) Repeat # 25ng 20ng 10ng 5ng 1ng CGG Summary • Short tandem repeats (STRs) impact human disease through variable length effects on gene expression, splicing, and translation. • STR expansions are linked to >30 human disorders, but their routine analysis has often been thwarted by the lack of fast, simple, and accurate assay systems. • We show that STRs in FMR1, C9orf72, TOMM40, DMPK, and HTT can be readily and reliably genotyped using on-market and prototype AmplideX ® PCR/CE reagents with the SeqStudio ™ Genetic Analyzer* (Thermo Fisher). Conclusions • When coupled with AmplideX ® PCR chemistries, the SeqStudio* platform provides versatile, sensitive, and accurate sizing of mono-, tri-, and hexa- nucleotide repeat expansions. • The flexibility of both the PCR technology and the SeqStudio* CE to resolve multiple AT- and GC-rich STRs, as well as CNVs such as SMN1, suggests that a broad range of markers and gene targets can be routinely analyzed using these methods. • These technologies can accelerate the development and adoption of STR-based assays for clinical research and future molecular diagnostics. Generalization of an Assay System using Repeat-primed PCR and Capillary Electrophoresis to Resolve Multiple AT- and GC-rich Short Tandem Repeats Associated with Neurological Diseases Gary J Latham 1 , Charles Redmond 1 , Kim Nicholson 1 , Errin Lagow 1 , Jeff Wallace 2 , Edgar Schreiber 2 , Scott Higdon 2 , and Eran Bram 1 1 Asuragen, Inc., Austin, TX USA 2 Genetic Sciences Division, Thermo Fisher Scientific, Inc., South San Francisco, CA USA Introduction DNA repeat sequences constitute roughly 50% of the human genome. Short tandem repeats (STRs) are a significant subclass of repeats that are associated with >30 known Mendelian disorders. The properties of these expanded repetitive sequences, however, challenge conventional PCR and associated analysis methods. The SeqStudio Genetic Analyzer* (Thermo Fisher) enables flexible, streamlined, and economical DNA fragment analysis using a universal polymer and cartridge-based system. Here, we describe the analytical performance of a suite of repeat- primed (RP) PCR assays on the SeqStudio Genetic Analyzer*. We also demonstrate the flexibility of the SeqStudio* to support copy number quantification using prototype AmplideX ® PCR/CE SMN1 reagents**. Materials and Methods Mono-, tri-, and hexa- nucleotide repeat expansions from STR disease markers, including TOMM40, FMR1, DMPK, HTT and C9orf72, as well as the AT-rich SMA-associated SMN1 exon 7 locus, were amplified from clinically-derived genomic DNA samples and cell-line materials (n=55) using the AmplideX ® PCR/CE FMR1 kit (CE-IVD), or other corresponding AmplideX ® PCR/CE kits (C9orf72, TOMM40, DMPK)* and prototype (HTT, SMN1)** reagents (Asuragen). FAM-labeled amplicons were resolved on the SeqStudio Genetic Analyzer* (Thermo Fisher) and compared with the 3500 Genetic Analyzer* (Thermo Fisher). SeqStudio* run settings were optimized across a range of injection and run conditions while assessing STR sizing accuracy, resolution, and sensitivity, along with copy number quantification for SMN1. *For Research Use Only, Not for Use in Diagnostic Procedures **This product is under development. Future availability and performance to be determined. Disclosures: CR, KN, EL, EB, and GJL have stock/stock options in and/or are employees of Asuragen, Inc. JW, ES, and SH have stock/stock options in and/or are employees of Thermo Fisher Scientific, Inc. Presented at ESHG 2018 - P14.039C Table 1. Comparison of General and AmplideX ® -specific Platform Specifications for the 3500* and SeqStudio Genetic Analyzers*. Figure 2. Representative SeqStudio* CE Profiles Generated using Commercially-available AmplideX ® PCR/CE Kits. A) FMR1 CGG repeat alleles ranging from 18 to >200 were amplified from the AmplideX ® FMR1 Control. B) A C9orf72 G 4 C 2 expansion in Coriell sample ND11081. The assay* also telegraphs 3’ sequence variations (inset: Compare top sample ND06769 vs. lower ND12947). C) AmplideX ® PCR/CE TOMM40 reagents* show single bp size resolution of gene-specific PCR products (Coriell samples NG09714 and ASGN005). Run times could be reduced to 20 min for TOMM40 polyT PCR products. Of note, different PCR/CE assays could be analyzed on the same CE plate using discrete or shared run conditions to harmonize and simplify batch run efficiency. Figure 5. Representative SeqStudio* Electropherograms Generated using using AmplideX ® PCR/CE DMPK* and prototype HTT** Reagents. A) Identification of a 25/~1100 CTG expansion in DMPK. Sample courtesy of Greenwood Genetics Center. B) A 15/70 CAG repeat expansion in HTT (Coriell sample NA13509) detected using a prototype 2-primer RP-PCR/CE assay. Figure 6. Quantitative Analysis of SMN1 Copy Number using Prototype AmplideX ® PCR/CE SMN1 Reagents** on the SeqStudio* Platform. Representative results from four gDNA samples reflecting 0-3 SMN1 copies were accurately genotyped across a 5-fold input dilution series using SeqStudio* relative fluorescence levels. Figure 4. Tunable Resolution and Sensitivity Demonstrated with Expanded C9orf72 Hexanucleotide Repeats. Run voltage on the SeqStudio* is a “tunable knob” for optimizing assay-specific variables such as resolution and peak detection sensitivity. Lower voltages extend the resolution of large fragments (top), whereas higher voltage reduces resolution and compounds the “peak pile-up” to enhance assay sensitivity (bottom). Optimized run conditions could extend the repeat resolution range by as much as 50%. Figure 3. Samples Amplified using AmplideX ® PCR/CE FMR1 and C9orf72* Reagents can be Sensitively and Accurately Resolved on the SeqStudio*, Consistent with Results on the 3500* CE. PCR of 126 C9orf72 and 448 FMR1 alleles from clinically-derived gDNA samples were evaluated on both the SeqStudio* and the 3500* CE. A) Single-repeat concordance between SeqStudio* and 3500* platforms. B) Detection of a full mutation FMR1 minor allele down to at least 5% mass fraction. C) All alleles in the AmplideX ® FMR1 Control were detected down to 1 ng of gDNA input. Figure 1. Schematic Representation of Single-tube, Long-range, 3-primer RP-PCR Assay Designs. Gene-specific (GS; blue) and repeat- primed (RP; red) primers create a multiplicity of FAM-labeled (star) PCR products from repetitive DNA (orange sawtooth). When separated by CE, these products manifest distinct full-length GS peaks and a complementary RP peak profile with single-repeat frequency and resolution. Results Feature 3500* Series SeqStudio* Capillary array 8/24 4 Capillary length 36/50 28 (Onboard click-in cartridge) Polymer type POP4/POP6/POP7 POP1 (Onboard click-in cartridge) Buffer system Separate Onboard click-in cartridge UI Ancillary computer Touchscreen/Cloud/Ancillary computer Footprint Large Small AmplideX ® Kit Recommended Run Settings Injection voltage 2.5 kV 6 kV Injection time 20 sec 2 sec Run voltage 19.5 kV 6 kV Run time 2400 sec 3300 sec 5 50 100 200 CGG CE Gene-Specific Amplicon Peak Repeat-Primed PCR 5’ 3’ 2A 3A 2B 3B 2C 3C 3.0 2.0 1.0 0.0 GM10684 GM03814 GM14820 GM17117 0 1 2 3 Expected Cp# SMN1 Cp# 20ng 80ng 100ng Ref Ref SMN1 Ref SMN1 Ref SMN1 5A 5B #P14.039C
Transcript of Generalization of an Assay System using Repeat-primed PCR ...€¦ · SeqStudio Genetic Analyzer*...
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Repeat Signal Detection vs % Minor Allele in FMR1 Sensitivity Control
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Summary• Short tandem repeats (STRs) impact human
disease through variable length effects on gene expression, splicing, and translation.
• STR expansions are linked to >30 human disorders, but their routine analysis has often been thwarted by the lack of fast, simple, and accurate assay systems.
• We show that STRs in FMR1, C9orf72, TOMM40, DMPK, and HTT can be readily and reliably genotyped using on-market and prototype AmplideX® PCR/CE reagents with the SeqStudio™ Genetic Analyzer* (Thermo Fisher).
Conclusions• When coupled with AmplideX® PCR chemistries,
the SeqStudio* platform provides versatile, sensitive, and accurate sizing of mono-, tri-, and hexa- nucleotide repeat expansions.
• The flexibility of both the PCR technology and the SeqStudio* CE to resolve multiple AT- and GC-rich STRs, as well as CNVs such as SMN1, suggests that a broad range of markers and gene targets can be routinely analyzed using these methods.
• These technologies can accelerate the development and adoption of STR-based assays for clinical research and future molecular diagnostics.
Generalization of an Assay System using Repeat-primed PCR and Capillary Electrophoresis to Resolve Multiple AT- and GC-rich Short Tandem Repeats Associated with Neurological DiseasesGary J Latham1, Charles Redmond1, Kim Nicholson1, Errin Lagow1, Jeff Wallace2, Edgar Schreiber2, Scott Higdon2, and Eran Bram1
1Asuragen, Inc., Austin, TX USA 2Genetic Sciences Division, Thermo Fisher Scientific, Inc., South San Francisco, CA USA
Introduction
DNA repeat sequences constitute roughly 50% of the human genome. Short tandem repeats (STRs) are a significant subclass of repeats that are associated with >30 known Mendelian disorders. The properties of these expanded repetitive sequences, however, challenge conventional PCR and associated analysis methods. The SeqStudio Genetic Analyzer* (Thermo Fisher) enables flexible, streamlined, and economical DNA fragment analysis using a universal polymer and cartridge-based system. Here, we describe the analytical performance of a suite of repeat-primed (RP) PCR assays on the SeqStudio Genetic Analyzer*. We also demonstrate the flexibility of the SeqStudio* to support copy number quantification using prototype AmplideX® PCR/CE SMN1 reagents**.
Materials and Methods
Mono-, tri-, and hexa- nucleotide repeat expansions from STR disease markers, including TOMM40, FMR1, DMPK, HTT and C9orf72, as well as the AT-rich SMA-associated SMN1 exon 7 locus, were amplified from clinically-derived genomic DNA samples and cell-line materials (n=55) using the AmplideX® PCR/CE FMR1 kit (CE-IVD), or other corresponding AmplideX® PCR/CE kits (C9orf72, TOMM40, DMPK)* and prototype (HTT, SMN1)** reagents (Asuragen). FAM-labeled amplicons were resolved on the SeqStudio Genetic Analyzer* (Thermo Fisher) and compared with the 3500 Genetic Analyzer* (Thermo Fisher). SeqStudio* run settings were optimized across a range of injection and run conditions while assessing STR sizing accuracy, resolution, and sensitivity, along with copy number quantification for SMN1.
*For Research Use Only, Not for Use in Diagnostic Procedures**This product is under development. Future availability and performance to be determined.Disclosures: CR, KN, EL, EB, and GJL have stock/stock options in and/or are employees of Asuragen, Inc. JW, ES, and SH have stock/stock options in and/or are employees of Thermo Fisher Scientific, Inc. Presented at ESHG 2018 - P14.039C
Table 1. Comparison of General and AmplideX®-specific Platform Specifications for the 3500* and SeqStudio Genetic Analyzers*.
Figure 2. Representative SeqStudio* CE Profiles Generated using Commercially-available AmplideX® PCR/CE Kits. A) FMR1 CGG repeat alleles ranging from 18 to >200 were amplified from the AmplideX® FMR1 Control. B) A C9orf72 G4C2 expansion in Coriell sample ND11081. The assay* also telegraphs 3’ sequence variations (inset: Compare top sample ND06769 vs. lower ND12947). C) AmplideX® PCR/CE TOMM40 reagents* show single bp size resolution of gene-specific PCR products (Coriell samples NG09714 and ASGN005). Run times could be reduced to 20 min for TOMM40 polyT PCR products. Of note, different PCR/CE assays could be analyzed on the same CE plate using discrete or shared run conditions to harmonize and simplify batch run efficiency.
Figure 5. Representative SeqStudio* Electropherograms Generated using using AmplideX® PCR/CE DMPK* and prototype HTT** Reagents. A) Identification of a 25/~1100 CTG expansion in DMPK. Sample courtesy of Greenwood Genetics Center. B) A 15/70 CAG repeat expansion in HTT (Coriell sample NA13509) detected using a prototype 2-primer RP-PCR/CE assay.
Figure 6. Quantitative Analysis of SMN1 Copy Number using Prototype AmplideX® PCR/CE SMN1 Reagents** on the SeqStudio* Platform. Representative results from four gDNA samples reflecting 0-3 SMN1 copies were accurately genotyped across a 5-fold input dilution series using SeqStudio* relative fluorescence levels.
Figure 4. Tunable Resolution and Sensitivity Demonstrated with Expanded C9orf72 Hexanucleotide Repeats. Run voltage on the SeqStudio* is a “tunable knob” for optimizing assay-specific variables such as resolution and peak detection sensitivity. Lower voltages extend the resolution of large fragments (top), whereas higher voltage reduces resolution and compounds the “peak pile-up” to enhance assay sensitivity (bottom). Optimized run conditions could extend the repeat resolution range by as much as 50%.
Figure 3. Samples Amplified using AmplideX® PCR/CE FMR1 and C9orf72* Reagents can be Sensitively and Accurately Resolved on the SeqStudio*, Consistent with Results on the 3500* CE. PCR of 126 C9orf72 and 448 FMR1 alleles from clinically-derived gDNA samples were evaluated on both the SeqStudio* and the 3500* CE. A) Single-repeat concordance between SeqStudio* and 3500* platforms. B) Detection of a full mutation FMR1 minor allele down to at least 5% mass fraction. C) All alleles in the AmplideX® FMR1 Control were detected down to 1 ng of gDNA input.
Figure 1. Schematic Representation of Single-tube, Long-range, 3-primer RP-PCR Assay Designs. Gene-specific (GS; blue) and repeat-primed (RP; red) primers create a multiplicity of FAM-labeled (star) PCR products from repetitive DNA (orange sawtooth). When separated by CE, these products manifest distinct full-length GS peaks and a complementary RP peak profile with single-repeat frequency and resolution.
Results
Feature 3500* Series SeqStudio*
Capillary array 8/24 4Capillary length 36/50 28 (Onboard click-in cartridge)Polymer type POP4/POP6/POP7 POP1 (Onboard click-in cartridge)Buffer system Separate Onboard click-in cartridgeUI Ancillary computer Touchscreen/Cloud/Ancillary computerFootprint Large SmallAmplideX® Kit Recommended Run SettingsInjection voltage 2.5 kV 6 kVInjection time 20 sec 2 secRun voltage 19.5 kV 6 kVRun time 2400 sec 3300 sec
5 50 100 200 CGG
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#P14.039C
