FREQ-Seq: A Rapid, Cost-Effective, Sequencing-Based Method to Determine Allele Frequencies Directly from Mixed Populations

被引:35
作者
Chubiz, Lon M. [1 ]
Lee, Ming-Chun [1 ]
Delaney, Nigel F. [1 ]
Marx, Christopher J. [1 ,2 ]
机构
[1] Harvard Univ, Dept Organism & Evolutionary Biol, Cambridge, MA 02138 USA
[2] Harvard Univ, Fac Arts & Sci, Ctr Syst Biol, Cambridge, MA 02138 USA
基金
美国国家卫生研究院; 美国国家科学基金会;
关键词
TERM EXPERIMENTAL EVOLUTION; ESCHERICHIA-COLI; 10,000-GENERATION EXPERIMENT; BENEFICIAL MUTATIONS; CLONAL INTERFERENCE; POOLED DNA; ADAPTATION; METHYLOBACTERIUM; SELECTION; GROWTH;
D O I
10.1371/journal.pone.0047959
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
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页数:9
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