New Software for Detecting Somatic Mutations at Low Level in Sanger Sequencing Traces
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The Minor Variant Finder (MVF) software developed by Thermo Fisher Scientific detects and reports somatic mutations in Sanger sequencing traces with 95.3% sensitivity and 99.8% specificity, identifying variants as low as 5%. It features novel algorithms for calling minor variants, a user-friendly workflow, and allows for integration with Next Generation Sequencing (NGS) for orthogonal verification. The software supports various file formats for output and is secured for desktop use without internet connection.
New Software for Detecting Somatic Mutations at Low Level in Sanger Sequencing Traces
- 1. Minor Variant Finder New Software for Detecting Somatic Mutations at Low Level in Sanger Sequencing Tracesg g q g Edgar Schreiber, Harrison Leong, Stephan Berosik, Jeff Marks, Stephanie Schneider, Wallace George, Hardeep Sangha, Yoke Peng Lim, Evelyn Chan, Dennis Nagtalon, and Joel Colburn Thermo Fisher Scientific Genetic Analysis Solutions, 180 Oyster Point Boulevard , South San Francisco CA 94080 Abstract Component Requirements Easy Project Set Up: Auto-Assisted or Manual Trace Organizer Report Outputs : PDF CSV VCF We have developed software that detects and reports 5% minor variants in Sanger Sequencing traces at 95.3% sensitivity and 99.8% specificity. The software calls variants without prior knowledge of location and affords the advantages of Sanger sequencing, of robustness, low error rate, ease of use, human interpretable visual displays of the data, and low cost per sample and target. The software can confirm somatic variants found by NGS. Noise minimization and peak detection algorithms subtract the baseline noise in a control sample from a test sample, detect candidate minor variants, and confirm them in the complementary strand. To test the new algorithms, synthetic mixtures of minor alleles were prepared by combining DNAs containing known mutations in known ratios. Using standard protocols for fluorescent dye terminator Sanger Component Requirements Computer Windows™ computer with 2 GB hard disk space and a minimum of 4 GB memory; 8 GB recommended. Operating system Windows™ 7 SP1, 32‐bit or 64‐bit, or Windows™ 10 Pro, 64‐bit Browser Google™ Chrome™, Mozilla™ Firefox™, Microsoft™ Internet Explorer™ v.11, or Microsoft™ Edge The Minor Variant Finder Software runs in a web browser windows, but does not require connection to the internet in order to run. Data is secure on your desktop computer. Organize the trace files: Trace files can be organized in two ways: i) manually by setting up folders for amplicons and test specimen and dragging/dropping the appropriate y j p g p p Glossary for column “Origin”: Common: Variant was found by MVF & NGS. MVF: Variant was found by MVF only sequencing, twenty‐five different amplicons and nine different genes, including TP53, KRAS, BRAF, EGFR, FLT3, RB1, CDH1, ERBB2, and XYLT1, from cell line and FFPE DNA were sequenced on 3500, 3730, and 3130 Applied Biosystems® Genetic Analyzers. 632,452 base positions and 2334 total variant positions, spanning variants at 2.5% to 50%, were interrogated. The software achieved 95.3% sensitivity and 99.83% specificity for automated detection of 885 variants present at the 5% level in 238,179 high quality base positions. Sensitivity was 98.8% for variants between 7.5‐10%, 98.7% for variants between 12.5‐25%, and 100% for variants ≥ 25%. The software displays a noise‐minimized trace view to facilitate visual inspection to confirm candidate variants. Further, reference sequences with hg19 chromosomal locations and NGS vcf files can be imported and aligned with the Sanger sequencing data for orthogonal verification. Hyperlinks to dbSNP for known rsSNP The Minor Variant Finder software scans the traces of a quartet of: • normal control forward and reverse strands • test sample forward and reverse strands for the presence of variants that occur in matching (i.e. sequence complementary) positions on both strands. At least one normal control pair (fwd/rev) must be present. Many test sample pairs for the same amplicon can be analyzed in the same session. A project can encompass multiple amplicons and reference sequences. First steps: Download free demo version at www.thermofisher.com/mvf trace file pairs into these folders or ii) automated‐assisted This convenient feature requires a consistent sample naming convention such as: sample ID_amplicon ID_orientation (FWD or REV) MVF: Variant was found by MVF only. NGS: Variant was found by NGS only. variants are provided in the software. We have demonstrated that Minor Variant Finder calls 5% somatic variants with 95.3% sensitivity and 99.8% specificity in Sanger sequencing traces. Introduction A minor variant is a heterozygous allele in which the proportion of the secondary allele is less than a classical Mendelian germ line heterozygous variant of 50:50. Detecting minor variants has become 1) Create a project 2) Import ab1 trace files into project Immediate QC Check of Imported Trace Files For minor variant detection, it is essential to use sequencing trace files of high quality Standardizing the sample name format greatly facilitates the set up of complex projects with multiple amplicons. Clear Presentation of Results h l h f h 5% Limit of Detection with 95.3% Sensitivity 99.8% Specificity MVF Algorithm Performance g yg g essential to combating, managing, and advancing our understanding of cancer, inherited and infectious diseases, and the impact of heteroplasmy on mitochondrial diseases. We have developed Minor Variant Finder (MVF) desktop software for the de novo discovery of minor variants, as well as next generation sequencing confirmation (NGC) of minor variants. Key software features include: Novel algorithms for calling minor variants. Testing demonstrates a 5% limit of detection with 95.3% sensitivity and 99.8% specificity. Easy‐to‐use workflow. use sequencing trace files of high quality. Guidance for achieving high quality traces is provided in companion protocols. Quality threshold settings can be customized to The Results Overview shows a map of the amplicon(s), genome or amplicon coordinates, DNA sequence and the position of variant candidates for both NGS and Sanger data. Clicking on a variant will open a comprehensive, detailed Results Table. The Results Table lists the Algorithm Performance was tested on a large data set. Synthetic mixtures of minor alleles were prepared by combining DNAs containing known variants in known ratios, in which DNA was obtained from cell line DNA or FFPE cell line DNA. DNA was also extracted from lung tumor FFPE samples, where the percent minor variant was determined by sequencing on Ion Torrent™ Personal Genome Machine® (PGM™) next generation sequencing (NGS). 25 amplicons across 9 genes, TP53, KRAS, BRAF, EGFR, FLT3, RB1, CDH1, ERBB2, and XYLT1, were sequenced using BigDye® Terminator v3 1 or Electropherograms and results workflow to enable users to easily review and accept variant calls. Functionality to import the human hg19 chromosome reference and next‐generation sequencing variants file for the NGS confirmation workflow, if the computer is web‐enabled. Ability to align and compare results between Sanger sequencing and Next Generation Sequencing (NGS) for orthogonal verification. Hyperlinks to dbSNP for known rsSNP variants are provided in the software. Trace file Quality Check, with preset thresholds for quick quality check review. Capability to export results in a variety of formats from pdf reports, comma‐separated files, to industry standard formats like vcf. P j bli d h i Q y g meet individual experimental needs. Proven default settings are provided. The QC module in MVF contains all the trace viewing functionality of the popular, free Applied Bi t S S ft High trace score value (>40) and signal to noise ratio (>180) are recommended for reliable minor variant detection. details for all variant candidates and their Review status. A colored “Review Indicator” indicates the goodness for the variant call: Green means the algorithm is confident; Yellow and Red mean increasing levels of uncertainty requiring extra scrutiny by the user. Hyperlink to dbSNP if variant is a known SNV. sequenced using BigDye Terminator v3.1 or BigDye® Direct Sequencing kits on POP‐7™ polymer on 3500, 3500xL, 3730xL, or 3130xL Applied Biosystems® Genetic Analyzers. Characteristics of Test Data Set 632,452 Total Number of basecalls 2,334 total number of variant positions Test Data % Minor Variant # of Expected Variants # of Found Variants # of Quartets # of Basecalls in Used Range Sensitivity Specificity 2.50% 399 138 399 143899 34.6% 99.76% 5% 885 843 754 238179 95.3% 99.83% 7.5‐10% 578 571 491 170214 98.8% 99.80% 12 5 25% 302 298 200 49657 98 7% 99 79% Summary for sensitivity (in %) for detection of rare (minor) variants at known % level (x‐axis). Project management enabling data sharing. Novel classification and signal processing algorithms were developed to identify minor variants. The algorithms take advantage of the reproducibility of the peak pattern in the Sanger sequencing traces, when the same locus of interest is sequenced in different specimens. The baseline noise is also typically very similar between different traces, provided the different DNAs are sequenced i lt l i th t i l t i t t t Th l d l d The How: Detecting Minor Variants out of “Reproducible Noise” Biosystems Sequence Scanner software. Multiple Options for Reference Import - Import NGS vcf file Creating a reference sequence in text format is optional; by default the forward t l i d The algorithm also requires confirmation of the minor variant in both forward and reverse strands to minimize false positives. The candidate variants the algorithm finds can easily be reviewed and accepted in the six electropherogram view. The Control electropherograms are shown in the lowest panel. The Test electropherograms are shown in the middle panel. The algorithm‐generated Test trace with the baseline noise minimized and the candidate variant revealed (marked by the red arrow) is Reviewing and Confirming Candidate Variants 12.5‐25% 302 298 200 49657 98.7% 99.79% >25% 170 170 170 30503 100.0% 99.77% Summary • The Minor Variant Finder software detects and calls minor variants as low as 5% from Sanger sequencing traces (.ab1 files) at high sensitivity (95.3%) and specificity (99.8%). • The algorithm neutralizes background noise signal by comparison of test sample(s) and a normal control sample. Th l ith t t t l t h ith th b k d i t li dsimultaneously, using the same reagents, primer lots, instruments, etc. The newly developed algorithms subtract the baseline noise in the control sequence from the baseline in the test sequence to reveal and thereby identify minor variants. The algorithms generate a noise minimized trace, labeled ‘NSS Algorithm Generated’ trace in the software, to enable easy reviewing by the user. control sequence is used. Import of a reference sequence with chromosomal positions (GRCh37; hg19) enables a link to the dbSNP database if a known rsSNP is detected. It is required if a NGS vcf file is imported for a verification project. Multiple reference sequences (each up to 10 kb length) can be set up. with the baseline noise minimized and the candidate variant revealed (marked by the red arrow) is displayed in the top panel. In addition, the algorithm calculates the peak height of the minor variant as a percent of the primary plus secondary peak heights for both the forward and reverse traces (circled in red.) Highly divergent peak heights between the forward and the reverse traces suggest the candidate is a false positive. Note: Peak heights in Sanger sequencing are not quantitative, and hence the ratio between primary and secondary peak heights does not precisely reflect allele frequency. • The algorithm generates test electropherograms with the background noise neutralized. • The algorithm calculates the ratio of the minor peak height relative to the peak heights of the primary plus secondary peak heights, in both the forward and reverse strands. • The minor variant is detected on both the forward and reverse strands. • The minor variant candidates are presented for review and confirmation (accept/reject/comment) by the user in a high resolution viewer tool that enables deep scrutiny of traces. • Reviewed and user confirmed results are reported in pdf, csv and vcf file formats. • The software aids in verification of minor variant findings obtained by NGS by reading the vcf file and aligning Sanger results with the NGS results. • A hyperlink to dbSNP is provided for known variants (if reference is available with chromosomal positions) Reproducible peak pattern in the primary sequence is shown between the Test sample (middle) and the Control sample (lower). Baseline noise peak pattern is also reproducible between Test (middle) and Control (lower). The minor variant in the test sample is revealed (top panel) when the baseline noise in Control is subtracted from baseline noise in Test. A vcf file can be imported into the project for verification of NGS results. The MVF software automatically filters out all AF=0 listings in the vcf file. chromosomal positions). • Free demo version is available for download at www.thermofisher.com/mvf. • For Research Use Only – Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific, Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.