Repository of Tomato Genomics Resources, Department of Plant Sciences, University of Hyderabad, Hyderabad, India.
Plant Breeding and Genetics Laboratory, IAEA Seibersdorf Laboratories, Reaktorstrasse 1, Seibersdorf, Austria.
Plant J. 2017 Nov;92(3):495-508. doi: 10.1111/tpj.13654. Epub 2017 Sep 10.
The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next-generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty-five genes belonging to carotenoids and folate metabolism were PCR-amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600-bp amplicons were directly sequenced in a non-overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128-fold pooling. An evaluation of six different software programs (camba, crisp, gatk unified genotyper, lofreq, snver and vipr) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.
靶向基因中的突变的鉴定因靶向诱导基因组局部突变(Targeting Induced Local Lesions In Genomes,TILLING)的出现而大大简化,加速了动植物的功能基因组分析。下一代测序(Next-generation sequencing,NGS)逐渐取代经典的 TILLING 用于突变检测,因为它允许在短时间内分析大量的扩增子。我们使用 NGS 方法鉴定了经乙基磺酸乙酯(ethylmethane sulphonate,EMS)双重诱变的番茄(Solanum lycopersicum)群体中的突变。我们对 25 个属于类胡萝卜素和叶酸代谢的基因进行 PCR 扩增并筛选,以鉴定潜在有益的等位基因。为了提高效率,我们以非重叠的方式在 Illumina MiSeq 上直接对 600bp 的扩增子进行测序,无需在文库制备前进行片段化步骤。不同池化深度的比较表明,杂合突变可在 128 倍的池化深度下鉴定。对六种不同软件程序(camba、crisp、gatk unified genotyper、lofreq、snver 和 vipr)的评估表明,没有一种软件程序能够稳健地以高保真度预测突变。其中,crisp 和 camba 预测的突变假阴性率较低。通过仅考虑两种不同软件程序共同预测的突变,可以大大减少假阳性。对 23.47 Mb 的番茄基因组进行筛选,得到了 75 个预测的突变,其中 64 个通过 Sanger 测序得到了验证,平均突变密度为 1/367 Kb。我们的结果表明,NGS 结合多种变异检测工具可以减少假阳性,并显著提高突变发现率。