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FREQ-Seq: a rapid, cost-effective, sequencing-based method to determine allele frequencies directly from mixed populations.
- Source :
- PLoS ONE, Vol 7, Iss 10, p e47959 (2012)
- Publication Year :
- 2012
- Publisher :
- Public Library of Science (PLoS), 2012.
-
Abstract
- Understanding evolutionary dynamics within microbial populations requires the ability to accurately follow allele frequencies through time. Here we present a rapid, cost-effective method (FREQ-Seq) that leverages Illumina next-generation sequencing for localized, quantitative allele frequency detection. Analogous to RNA-Seq, FREQ-Seq relies upon counts from the >10(5) reads generated per locus per time-point to determine allele frequencies. Loci of interest are directly amplified from a mixed population via two rounds of PCR using inexpensive, user-designed oligonucleotides and a bar-coded bridging primer system that can be regenerated in-house. The resulting bar-coded PCR products contain the adapters needed for Illumina sequencing, eliminating further library preparation. We demonstrate the utility of FREQ-Seq by determining the order and dynamics of beneficial alleles that arose as a microbial population, founded with an engineered strain of Methylobacterium, evolved to grow on methanol. Quantifying allele frequencies with minimal bias down to 1% abundance allowed effective analysis of SNPs, small in-dels and insertions of transposable elements. Our data reveal large-scale clonal interference during the early stages of adaptation and illustrate the utility of FREQ-Seq as a cost-effective tool for tracking allele frequencies in populations.
Details
- Language :
- English
- ISSN :
- 19326203
- Volume :
- 7
- Issue :
- 10
- Database :
- Directory of Open Access Journals
- Journal :
- PLoS ONE
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.1b4d00acefe4c11a5ae059134f1b650
- Document Type :
- article
- Full Text :
- https://doi.org/10.1371/journal.pone.0047959