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NPJ Genom Med. 2018 Dec 18;3:34. doi: 10.1038/s41525-018-0072-5. eCollection 2018.

TAC-seq: targeted DNA and RNA sequencing for precise biomarker molecule counting.

Author information

1
1Competence Centre on Health Technologies, Tartu, Estonia.
2
2Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia.
3
3Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
4
4Institute of Computer Science, University of Tartu, Tartu, Estonia.
5
5Institute of Clinical Medicine, Department of Obstetrics and Gynaecology, University of Tartu, Tartu, Estonia.
6
6Estonian Genome Center, University of Tartu, Tartu, Estonia.
7
7Department of Chemistry and Biotechnology, School of Science, Tallinn University of Technology, Tallinn, Estonia.
8
8Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.
9
9Research Program of Molecular Neurology, Research Programs Unit, University of Helsinki, and Folkhälsan Institute of Genetics, Helsinki, Finland.
10
10School of Basic and Medical Biosciences, Guy's Hospital, King's College London, London, UK.
11
11Institute of Biomedicine and Translational Medicine, Department of Biomedicine, University of Tartu, Tartu, Estonia.
12
12Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
13
13Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland.
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Contributed equally

Abstract

Targeted next-generation sequencing (NGS) methods have become essential in medical research and diagnostics. In addition to NGS sensitivity and high-throughput capacity, precise biomolecule counting based on unique molecular identifier (UMI) has potential to increase biomolecule detection accuracy. Although UMIs are widely used in basic research its introduction to clinical assays is still in progress. Here, we present a robust and cost-effective TAC-seq (Targeted Allele Counting by sequencing) method that uses UMIs to estimate the original molecule counts of mRNAs, microRNAs, and cell-free DNA. We applied TAC-seq in three different clinical applications and compared the results with standard NGS. RNA samples extracted from human endometrial biopsies were analyzed using previously described 57 mRNA-based receptivity biomarkers and 49 selected microRNAs at different expression levels. Cell-free DNA aneuploidy testing was based on cell line (47,XX, +21) genomic DNA. TAC-seq mRNA profiling showed identical clustering results to transcriptome RNA sequencing, and microRNA detection demonstrated significant reduction in amplification bias, allowing to determine minor expression changes between different samples that remained undetermined by standard NGS. The mimicking experiment for cell-free DNA fetal aneuploidy analysis showed that TAC-seq can be applied to count highly fragmented DNA, detecting significant (p = 7.6 × 10-4) excess of chromosome 21 molecules at 10% fetal fraction level. Based on three proof-of-principle applications we demonstrate that TAC-seq is an accurate and highly potential biomarker profiling method for advanced medical research and diagnostics.

Conflict of interest statement

Kaarel Krjutškov, Mariann Koel, Juha Kere, and Andres Salumets declare that they have submitted an international patent application on the TAC-seq method. The other authors declare no competing interests.

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