Estimating UMI-Tag count: UMI-TnSeq protocol for identifying fitness-relevant genes in the PCB degrader Acidovorax sp. KKS102
- 1Department of Molecular and Chemical Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.
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.
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BibTeX
@article{ohtsubo2026estimating,
title = {{Estimating UMI-Tag count: UMI-TnSeq protocol for identifying fitness-relevant genes in the PCB degrader Acidovorax sp. KKS102}},
author = {Yoshiyuki Ohtsubo and Yutarou Hemmi and Shiho Abe and Shouta Nonoyama and Kouhei Kishida and Yuji Nagata},
journal = {DNA research : an international journal for rapid publication of reports on genes and genomes},
year = {2026},
month = oct,
doi = {10.1093/dnares/dsag017},
url = {https://europepmc.org/article/PMC/PMC13721135},
note = {CC BY},
}
RIS
TY - JOUR
TI - Estimating UMI-Tag count: UMI-TnSeq protocol for identifying fitness-relevant genes in the PCB degrader Acidovorax sp. KKS102
AU - Yoshiyuki Ohtsubo
AU - Yutarou Hemmi
AU - Shiho Abe
AU - Shouta Nonoyama
AU - Kouhei Kishida
AU - Yuji Nagata
PY - 2026
DA - 2026/10/01
JO - DNA research : an international journal for rapid publication of reports on genes and genomes
DO - 10.1093/dnares/dsag017
UR - https://europepmc.org/article/PMC/PMC13721135
AB - 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.
SN - 1340-2838
SN - 1756-1663
ER -