title: Limitations and challenges of genetic barcode quantification.
abstract: Genetic barcodes are increasingly used to track individual cells and to quantitatively assess their clonal contributions over time. Although barcode quantification relies entirely on counting sequencing reads, detailed studies about the method's accuracy are still limited. We report on a systematic investigation of the relation between barcode abundance and resulting read counts after amplification and sequencing using cell-mixtures that contain barcodes with known frequencies ("miniBulks"). We evaluated the influence of protocol modifications to identify potential sources of error and elucidate possible limitations of the quantification approach. Based on these findings we designed an advanced barcode construct (BC32) to improved barcode calling and quantification, and to ensure a sensitive detection of even highly diluted barcodes. Our results emphasize the importance of using curated barcode libraries to obtain interpretable quantitative data and underline the need for rigorous analyses of any utilized barcode library in terms of reliability and reproducibility.
authors.affiliation: Institute for Medical Informatics and Biometry, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, 01307 Dresden, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Thielecke
authors.affiliation: Research Dept. Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Aranyossy
authors.affiliation: Deep Sequencing Group SFB 655, Biotechnology Center, Technische Universität Dresden, 01307 Dresden, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Dahl
authors.affiliation: Institute of Molecular Medicine and Stem Cell Aging, University of Ulm, Albert-Einstein-Allee 11c, D-89081 Ulm, Germany.
authors.forename: Rajiv
authors.initials: R
authors.lastname: Tiwari
authors.affiliation: Institute for Medical Informatics and Biometry, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, 01307 Dresden, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Roeder
authors.affiliation: Institute of Molecular Medicine and Stem Cell Aging, University of Ulm, Albert-Einstein-Allee 11c, D-89081 Ulm, Germany.|Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH, USA.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Geiger
authors.affiliation: Research Dept. Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Fehse
authors.affiliation: Institute for Medical Informatics and Biometry, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, 01307 Dresden, Germany.
authors.forename: Ingmar
authors.initials: I
authors.lastname: Glauche
authors.affiliation: Research Dept. Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Cornils
mesh_terms: D002452:Cell Count / Q000379:methods*; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA