Precise measurement of the fitness effects of spontaneous mutations by droplet digital PCR in Burkholderia cenocepacia.

Autor:	Rana A; Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, Charlotte, NC 28223, USA., Patton D; Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, Charlotte, NC 28223, USA., Turner NT; Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, Charlotte, NC 28223, USA., Dillon MM; Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S3B2, Canada., Cooper VS; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA., Sung W; Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, Charlotte, NC 28223, USA.
Jazyk:	angličtina
Zdroj:	Genetics [Genetics] 2021 Oct 02; Vol. 219 (2).
DOI:	10.1093/genetics/iyab117
Abstrakt:	Understanding how mutations affect survivability is a key component to knowing how organisms and complex traits evolve. However, most mutations have a minor effect on fitness and these effects are difficult to resolve using traditional molecular techniques. Therefore, there is a dire need for more accurate and precise fitness measurements methods. Here, we measured the fitness effects in Burkholderia cenocepacia HI2424 mutation accumulation (MA) lines using droplet-digital polymerase chain reaction (ddPCR). Overall, the fitness measurements from ddPCR-MA are correlated positively with fitness measurements derived from traditional phenotypic marker assays (r = 0.297, P = 0.05), but showed some differences. First, ddPCR had significantly lower measurement variance in fitness (F = 3.78, P < 2.6 × 10-13) in control experiments. Second, the mean fitness from ddPCR-MA measurements were significantly lower than phenotypic marker assays (-0.0041 vs -0.0071, P = 0.006). Consistent with phenotypic marker assays, ddPCR-MA measurements observed multiple (27/43) lineages that significantly deviated from mean fitness, suggesting that a majority of the mutations are neutral or slightly deleterious and intermixed with a few mutations that have extremely large effects. Of these mutations, we found a significant excess of mutations within DNA excinuclease and Lys R transcriptional regulators that have extreme deleterious and beneficial effects, indicating that modifications to transcription and replication may have a strong effect on organismal fitness. This study demonstrates the power of ddPCR as a ubiquitous method for high-throughput fitness measurements in both DNA- and RNA-based organisms regardless of cell type or physiology. (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
Databáze:	MEDLINE
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