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.

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent

“…1 cells, we transduced these cells (18-24 hours) with H/F-LVs encoding for a 32-bp-long barcode library 54 at low vector doses (MOI, 5) without cytokine stimulation before injection into NSG mice. After the mice had been reconstituted for 4 months, we isolated different hematopoietic lineages in the BM, spleen, and thymocytes and identified the barcodes present in the different lineages ( Figure 6A).…”

Clonal competition in BcrAbl-driven leukemia: how transplantations can accelerate clonal conversion

“…The BC32-construct as described in Thielecke et al [23] was introduced into the viral backbone by Gibson assembly [24]. The plasmid library was used for production of eco-pseudotyped retroviral particles as described [25].…”

DNA barcode-guided lentiviral CRISPRa tool to trace and isolate individual clonal lineages in heterogeneous cancer cell populations

“…Then, the barcode sequences were filtered according to the minimal (>20) and average (>30) Phred quality scores, and the arcode frequencies were calculated. To account for PCR and NGS errors we adopted an algorithm similar to previously reported 13,48 . Briefly, the algorithm starts by picking a barcode with the lowest frequency and calculates Hamming distances to all the other barcodes.…”