A sequencing-based method for quantifying gene-deletion mutants of bacteria in the intracellular environment
Advancements in next-generation sequencing (NGS) have significantly accelerated the development of innovative methodologies in microbiological research. In this study, we present a novel method to quantify the net survival of gene-deletion mutants within the intracellular environment. Based on stand...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-01-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1487724/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832583815206273024 |
|---|---|
| author | Xiao Fei Xiao Fei Zengzhi Yuan Zengzhi Yuan Sandra Marina Wellner Yibing Ma John Elmerdahl Olsen |
| author_facet | Xiao Fei Xiao Fei Zengzhi Yuan Zengzhi Yuan Sandra Marina Wellner Yibing Ma John Elmerdahl Olsen |
| author_sort | Xiao Fei |
| collection | DOAJ |
| description | Advancements in next-generation sequencing (NGS) have significantly accelerated the development of innovative methodologies in microbiological research. In this study, we present a novel method to quantify the net survival of gene-deletion mutants within the intracellular environment. Based on standardized Illumina short-read sequencing of genomic DNA, the method eliminates the need for specific selective markers on each deletion mutant. For validation, the method was shown to accurately quantify mutants in spiked pools of mixed mutants, showing no statistically significant differences compared to the expected values based on CFU determination (p > 0.05). Further, the method was used to quantify mutants of S. Gallinarum in macrophages. Six mutants and one control strain were mixed in a pool and allowed to infect HD11 cells for 2 h. The results align with prior research results, providing evidence of the feasibility of mixed mutant infections in functional gene identification. Notably, the simplicity and standardization of the method, rooted in standard whole-genome sequencing protocols, make it easily implementable across various laboratories. |
| format | Article |
| id | doaj-art-cca3af5cf7bf4eab8f9d83f5e1f57dbb |
| institution | Kabale University |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj-art-cca3af5cf7bf4eab8f9d83f5e1f57dbb2025-01-28T06:41:00ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-01-011510.3389/fmicb.2024.14877241487724A sequencing-based method for quantifying gene-deletion mutants of bacteria in the intracellular environmentXiao Fei0Xiao Fei1Zengzhi Yuan2Zengzhi Yuan3Sandra Marina Wellner4Yibing Ma5John Elmerdahl Olsen6Key Laboratory of Animal Pathogen Infection and Immunology of Fujian Province, College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou, ChinaDepartment of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DenmarkTianjin Key Laboratory of Animal and Plant Resistance, Tianjin, ChinaCollege of Life Sciences, Tianjin Normal University, Tianjin, ChinaDepartment of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DenmarkDepartment of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DenmarkDepartment of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DenmarkAdvancements in next-generation sequencing (NGS) have significantly accelerated the development of innovative methodologies in microbiological research. In this study, we present a novel method to quantify the net survival of gene-deletion mutants within the intracellular environment. Based on standardized Illumina short-read sequencing of genomic DNA, the method eliminates the need for specific selective markers on each deletion mutant. For validation, the method was shown to accurately quantify mutants in spiked pools of mixed mutants, showing no statistically significant differences compared to the expected values based on CFU determination (p > 0.05). Further, the method was used to quantify mutants of S. Gallinarum in macrophages. Six mutants and one control strain were mixed in a pool and allowed to infect HD11 cells for 2 h. The results align with prior research results, providing evidence of the feasibility of mixed mutant infections in functional gene identification. Notably, the simplicity and standardization of the method, rooted in standard whole-genome sequencing protocols, make it easily implementable across various laboratories.https://www.frontiersin.org/articles/10.3389/fmicb.2024.1487724/fullnext-generation sequencingintracellular survivalgene-deletion mutantsSalmonellabacteria |
| spellingShingle | Xiao Fei Xiao Fei Zengzhi Yuan Zengzhi Yuan Sandra Marina Wellner Yibing Ma John Elmerdahl Olsen A sequencing-based method for quantifying gene-deletion mutants of bacteria in the intracellular environment Frontiers in Microbiology next-generation sequencing intracellular survival gene-deletion mutants Salmonella bacteria |
| title | A sequencing-based method for quantifying gene-deletion mutants of bacteria in the intracellular environment |
| title_full | A sequencing-based method for quantifying gene-deletion mutants of bacteria in the intracellular environment |
| title_fullStr | A sequencing-based method for quantifying gene-deletion mutants of bacteria in the intracellular environment |
| title_full_unstemmed | A sequencing-based method for quantifying gene-deletion mutants of bacteria in the intracellular environment |
| title_short | A sequencing-based method for quantifying gene-deletion mutants of bacteria in the intracellular environment |
| title_sort | sequencing based method for quantifying gene deletion mutants of bacteria in the intracellular environment |
| topic | next-generation sequencing intracellular survival gene-deletion mutants Salmonella bacteria |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1487724/full |
| work_keys_str_mv | AT xiaofei asequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT xiaofei asequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT zengzhiyuan asequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT zengzhiyuan asequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT sandramarinawellner asequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT yibingma asequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT johnelmerdahlolsen asequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT xiaofei sequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT xiaofei sequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT zengzhiyuan sequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT zengzhiyuan sequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT sandramarinawellner sequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT yibingma sequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment AT johnelmerdahlolsen sequencingbasedmethodforquantifyinggenedeletionmutantsofbacteriaintheintracellularenvironment |