Limitations and challenges of genetic barcode quantification
#MMPMID28256524
Thielecke L
; Aranyossy T
; Dahl A
; Tiwari R
; Roeder I
; Geiger H
; Fehse B
; Glauche I
; Cornils K
Sci Rep
2017[Mar]; 7
(?): 43249
PMID28256524
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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.
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