qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing
Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify nonneutral mutations by selection or counterselection, the identification of neutral mutations in a heterogeneous population usually requires expensive and time-...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Association for the Advancement of Science (AAAS)
2023-01-01
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| Series: | BioDesign Research |
| Online Access: | https://spj.science.org/doi/10.34133/bdr.0007 |
| _version_ | 1797279818670145536 |
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| author | Satya Prakash Adrian Racovita Teresa Petrucci Roberto Galizi Alfonso Jaramillo |
| author_facet | Satya Prakash Adrian Racovita Teresa Petrucci Roberto Galizi Alfonso Jaramillo |
| author_sort | Satya Prakash |
| collection | DOAJ |
| description | Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify nonneutral mutations by selection or counterselection, the identification of neutral mutations in a heterogeneous population usually requires expensive and time-consuming methods such as quantitative or droplet polymerase chain reaction and high-throughput sequencing. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counterselection. We propose a novel method, which we called qSanger, to quantify DNA using amplitude ratios of aligned electropherogram peaks from mixed Sanger sequencing reads. Plasmids expressing enhanced green fluorescent protein and mCherry fluorescent markers were used to validate qSanger both in vitro and in cotransformed Escherichia coli via quantitative polymerase chain reaction and fluorescence quantifications. We show that qSanger allows the quantification of genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with substantial reduction of labor and costs compared to canonical approaches. |
| first_indexed | 2024-03-07T16:31:27Z |
| format | Article |
| id | doaj.art-5344c33e13ed4f7098ff639550a5e6ab |
| institution | Directory Open Access Journal |
| issn | 2693-1257 |
| language | English |
| last_indexed | 2024-03-07T16:31:27Z |
| publishDate | 2023-01-01 |
| publisher | American Association for the Advancement of Science (AAAS) |
| record_format | Article |
| series | BioDesign Research |
| spelling | doaj.art-5344c33e13ed4f7098ff639550a5e6ab2024-03-03T10:12:05ZengAmerican Association for the Advancement of Science (AAAS)BioDesign Research2693-12572023-01-01510.34133/bdr.0007qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger SequencingSatya Prakash0Adrian Racovita1Teresa Petrucci2Roberto Galizi3Alfonso Jaramillo4School of Life Sciences, University of Warwick, Coventry, UK.De Novo Synthetic Biology Lab, I2SysBio, CSIC-University of Valencia, Paterna, Spain.Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy.Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Keele, UK.School of Life Sciences, University of Warwick, Coventry, UK.Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify nonneutral mutations by selection or counterselection, the identification of neutral mutations in a heterogeneous population usually requires expensive and time-consuming methods such as quantitative or droplet polymerase chain reaction and high-throughput sequencing. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counterselection. We propose a novel method, which we called qSanger, to quantify DNA using amplitude ratios of aligned electropherogram peaks from mixed Sanger sequencing reads. Plasmids expressing enhanced green fluorescent protein and mCherry fluorescent markers were used to validate qSanger both in vitro and in cotransformed Escherichia coli via quantitative polymerase chain reaction and fluorescence quantifications. We show that qSanger allows the quantification of genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with substantial reduction of labor and costs compared to canonical approaches.https://spj.science.org/doi/10.34133/bdr.0007 |
| spellingShingle | Satya Prakash Adrian Racovita Teresa Petrucci Roberto Galizi Alfonso Jaramillo qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing BioDesign Research |
| title | qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing |
| title_full | qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing |
| title_fullStr | qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing |
| title_full_unstemmed | qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing |
| title_short | qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing |
| title_sort | qsanger quantification of genetic variants in bacterial cultures by sanger sequencing |
| url | https://spj.science.org/doi/10.34133/bdr.0007 |
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