title: FREQ-Seq: a rapid, cost-effective, sequencing-based method to determine allele frequencies directly from mixed populations.
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.
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Lon M
authors.initials: LM
authors.lastname: Chubiz
authors.forename: Ming-Chun
authors.initials: MC
authors.lastname: Lee
authors.forename: Nigel F
authors.initials: NF
authors.lastname: Delaney
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Marx
mesh_terms: D000483:Alleles; D002138:Calibration; D004251:DNA Transposable Elements; D019143:Evolution, Molecular; D005787:Gene Frequency; D015723:Gene Library; D005798:Genes, Bacterial; D005838:Genotype; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D054643:INDEL Mutation; D020601:Methylobacterium extorquens / Q000235:genetics* / Q000254:growth & development; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA
chemical_list: D004251:DNA Transposable Elements