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24 | OCTOBER 1, 2016 | GENengnews.com | Genetic Engineering & Biotechnology News Kristine Angevine, Edward Jan, Matt Carroll, Jinglan Zhang, Hongzheng Dai, Richard Yim, and Shawn Quinn Research in next-generation sequencing (NGS) is rapidly evolving, especially in sev- eral areas of cancer research that rely on de- tection of mutations. In contrast to germline mutations, somatic mutations may be present at low frequencies within an excess of DNA from normal cells. Examination of cell-free DNA (cfDNA) is a non-invasive method to study the presence of circulating tumor DNA, which contains somatic mutations. Thus, the ability to de- tect low-frequency alleles in cfDNA is crucial to many research applications including the tracking of tumor recurrence and character- ization of tumor clones. NGS methods suffer from technical draw- backs that pose challenges for low-frequency mutation detection. Most commonly, base er- rors are generated and accumulated through the library preparation and sequencing pro- cess, making it impossible to accurately re- construct the sequence of the original mol- ecules and differentiate real mutations from false positives introduced during amplifica- tion, sequencing, and base-calling steps. As a result, it is difficult to identify true mutations, because they are hidden by false base calls, also known as false positives. To overcome these challenges, short, ran- dom DNA sequences, named unique molecu- lar tags (UMTs), can be used during library preparation to label input DNA fragments. This method allows for the removal of back- ground errors during data processing so true mutations can be determined. Rubicon Ge- nomics' ThruPLEX Tag-seq kit is an Illumi- na ® NGS library preparation kit that contains over 16 million UMTs to provide confident mutation calls and has a single-tube, three- step workflow. ThruPLEX Tag-seq Workflow In the first step, DNA fragments are end- repaired in a highly efficient process. Follow- ing repair, proprietary ThruPLEX stem-loop adapters containing UMTs are ligated to DNA fragments and extended. The UMTs al- low individual DNA fragments to be tracked throughout the library preparation and data analysis process. In the final reaction, index- ing primers containing Illumina P5 and P7 sequences are used to complete the library structure and amplify the library fragments. Sample indexes—either single or dual—are incorporated to allow pooling and multiplex- Our distinguished scientific committee invites you to join them: Registration is now open for the upcoming 10th HIC/RPC Hydrophobic Bioprocessing Conference Jürgen Hubbuch Karlsruhe Institute of Tech. Karlsruhe, Germany Shuichi Yamamoto Yamaguchi University Yamaguchi, Japan Ron Bates Bristol-Myers Squibb Syracuse, New York Alois Jungbauer Univ. of Natural Resources & Life Sciences Vienna, Austria Eggert Brekkan GE Healthcare Uppsala Sweden J. Kevin O'Donnell Tosoh Bioscience LLC King of Prussia, Pennsylvania Shekhar Garde Rensselaer Polytechnic Inst. Troy, New York Arne Staby Novo Nordisk A/S Gentofte, Denmark Media partner: www.hic-rpc.org Be a part of this exciting technical conference! Register and submit your abstract today at: Committee Chair: Todd Przybycien Carnegie Mellon University Pittsburgh, Pennsylvania ThruPLEX® Tag-seq Kit Reduces False Positives for Discovery of True Mutations Detect Rare Alleles in NGS with Molecular Tags OMICS Tutorial Figure 1. Determining true mutations. The ThruPLEX Tag-seq Kit uses UMTs to label the initial molecule containing the mutation (green dot) at both ends. After library preparation and sequencing, PCR artifacts (blue dot) and sequencing errors (red dot) can be distinguished bioinformatically from the initial molecule, allowing the detection of true mutations and reducing the number of false positives. Table 1. Variant frequency detection. Horizon Multiplex I cfDNA Reference Standards (Horizon HD780) were used as is or titrated using the wild-type reference standard to generate samples at additional allele frequencies. Variants were detected at their expected frequencies with high sensitivity and specificity. Figure 2. Background reduction using UMT. Data from the same sample was processed without (A) or with UMTs (B). (A) In conventional library prep, true mutations (yellow dot) can be difficult to detect due to background errors (gray dot). (B) With the use of UMTs, the background is significantly reduced and the true mutation can be detected. (C) The signal-to-noise ratio is significantly improved at various allele frequencies between conventional library preparation (gray) and library preparation containing UMTs (yellow). A B C