FREQ-Seq: a rapid, cost-effective, sequencing-based method to determine allele frequencies directly from mixed populations.
FREQ-Seq: a rapid, cost-effective, sequencing-based method to determine allele frequencies directly from mixed populations.
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FREQ-Seq：一种快速、经济高效、基于测序的方法，可直接从混合群体中确定等位基因频率。
DOI：
10.1371/journal.pone.0047959
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Marx CJ
中科院分区：
文献类型：
--
作者：
Chubiz LM;Lee MC;Delaney NF;Marx CJ
了解微生物种群内的进化动态需要能够准确跟踪随时间变化的等位基因频率。在这里，我们提出了一种快速、经济高效的方法 (FREQ-Seq)，该方法利用 Illumina 新一代测序进行局部、定量等位基因频率检测。与 RNA-Seq 类似，FREQ-Seq 依赖于每个时间点每个位点生成的 >105 个读数的计数来确定等位基因频率。使用廉价的、用户设计的寡核苷酸和可内部再生的条形码桥接引物系统，通过两轮 PCR 直接从混合群体中扩增感兴趣的位点。生成的条形码 PCR 产品包含 Illumina 测序所需的接头，无需进一步的文库制备。我们通过确定有益等位基因的顺序和动态来证明 FREQ-Seq 的实用性，这些等位基因是由甲基杆菌工程菌株建立的微生物群体产生的，并进化为在甲醇中生长。以低至 1% 丰度的最小偏差量化等位基因频率，可以有效分析 SNP、小插入缺失和转座元件插入。我们的数据揭示了适应早期阶段的大规模克隆干扰，并说明了 FREQ-Seq 作为跟踪人群等位基因频率的经济高效工具的效用。
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 >105 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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影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
2.9
作者：
LENSKI, RE;ROSE, MR;TADLER, SC
通讯作者：
TADLER, SC
影响因子：
4.4
作者：
Kirov, G;Nikolov, I;O'Donovan, MC
通讯作者：
O'Donovan, MC
DOI：
10.1073/pnas.91.15.6808
发表时间：
1994-07-19
影响因子：
11.1
作者：
LENSKI, RE;TRAVISANO, M
通讯作者：
TRAVISANO, M
影响因子：
5.3
作者：
Norton, N;Williams, NM;O'Donovan, MC
通讯作者：
O'Donovan, MC