Mar 4, 2017

Limitations and challenges of genetic barcode quantification

Scientific Reports
Lars ThieleckeKerstin Cornils


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.

Mentioned in this Paper

HEK293 Cells
Nucleic Acid Amplification Tests
Nucleic Acid Sequencing
Gene Amplification Technique
Cell Density
Sequence Determinations, DNA
DNA Barcoding, Taxonomic

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