Estimating UMI-Tag count: UMI-TnSeq protocol for identifying fitness-relevant genes in the PCB degrader Acidovorax sp. KKS102
Year: 2026
Type: article
Abstract: We developed a novel UMI-TnSeq (unique molecular identifier TnSeq) protocol to enhance the accuracy of quantifying the relative abundance of Tn mutants within a library. This improved approach includes custom-designed Tn-cassettes embedded with Illumina sequencing-compatible sequences and a unique adapter ligation protocol using reverse transcriptase to incorporate UMIs efficiently. Applying this refined UMI-TnSeq to the PCB/biphenyl-degrading Acidovorax sp. strain SA17 enabled us to identify genetic alterations that increased relative abundance in biphenyl media, including disruption of relA and transposon insertions predicted to increase the expression of a gene cluster encoding benzoate transporters. In addition, we introduced 2 novel metrics: the Gini coefficient and the "N-estimate." The Gini coefficient tracks selection progression by measuring shifts toward fitter mutants, while the N-estimate, calculated via model parameter fitting, evaluates UMI diversity and enables calculation of the "Detection Completeness Value." These mathematical improvements are broadly applicable and beneficial for other UMI-based studies.
Source: DNA Research
Institution: Tohoku University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,400
Award: G1183293057
OpenAlex ID: W7224946183