Data from: High throughput method for analysis of repeat number for 28 phase variable loci of Campylobacter jejuni strain NCTC11168

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method—the 28-locus-CJ11168 PV-analysis assay—for rapidly determining ON/OFF states of 28 of these phase-variable loci in a large number of individual colonies from C. jejuni strain NCTC11168. The method combines a series of multiplex PCR assays with a fragment analysis assay and automated extraction of fragment length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. Application of this method to analysis of the 28 polyG/polyC tracts in 90 C. jejuni colonies detected a 2.5-fold increase in slippage products as tracts lengthened from G8 to G11 but no difference between tracts of similar length indicating that flanking sequence does not influence slippage rates. Comparison of this observed slippage to previously measured mutation rates for G8 and G11 tracts in C. jejuni indicates that PCR amplification of a DNA sample will over-estimate phase variation frequencies by 20-35-fold. An important output of the 28-locus-CJ11168 PV-analysis assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species.简单序列重复序列区域（simple sequence repeat tracts）内的突变，是空肠弯曲杆菌（Campylobacter jejuni）在内的多种细菌物种相变（phase variation）的核心机制。位于可读框（reading frame）内的重复序列区域的重复单元数量变化，可使基因表达在“开启（ON）”与“关闭（OFF）”状态间以高频发生可逆切换。空肠弯曲杆菌NCTC11168菌株的基因组中，存在29个含有7个及以上重复单元的多聚G/多聚C序列区域（polyG/polyC tracts）。本方案详述了一种名为“28位点-CJ11168相变分析测定法（28-locus-CJ11168 PV-analysis assay）”的方法，可快速检测空肠弯曲杆菌NCTC11168菌株的28个相变可变位点的开启/关闭状态，适用于大批量单个菌落样本。该方法将多重聚合酶链式反应（multiplex PCR）系列实验、片段分析实验与片段长度、重复单元数量及表达状态的自动化提取流程相结合。这种高通量多重分析方法，可用于检测不同时间点种群内部及种群间相变状态的动态变化，并可用于探究相变对细菌适应不同选择压力的影响。将该方法应用于90个空肠弯曲杆菌菌落的28个多聚G/多聚C序列区域分析时，研究发现当序列区域从G8延伸至G11时，滑动错配（slippage）产物数量增加2.5倍；而长度相近的序列区域之间并无显著差异，这表明侧翼序列不会影响滑动错配速率。将本次观测到的滑动错配现象与此前测得的空肠弯曲杆菌G8和G11序列区域的突变率进行对比后发现，DNA样本的聚合酶链式反应扩增会将相变频率高估20至35倍。“28位点-CJ11168相变分析测定法”的一项重要产出结果，是其他方法无法获取的组合式表达状态信息。该方法可经调整后，用于其他空肠弯曲杆菌菌株以及多种其他细菌物种的相变分析。