Vanessa cardui, whole-genome resequencing for genotyping of ivory lncRNA crispants. Vanessa cardui

Genotyping by resequencing of butterfly samples including crispants generated by dual targeting of the ivory lncRNA exons 1 and 2. Syncitial embryos collected from Vanessa cardui were injected between 45 min and 2 h AEL, using a combination of two sgRNAs targeting the ivory lncRNA first or second exon. Adult crispants displaying large mutant clones in the G0 generation were pooled in cages of 4-15 butterflies for random in-crossing. G1 individuals were randomly in-crossed again in cages of 4-15 butterflies to generate G2 individuals. DNA from V. cardui butterflies displaying strong [ivory] phenotypes was then extracted from adult thoraces. Sequencing libraries (PE 150) were generated and sequenced in an Illumina NovaSeq6000 S4 Flow cell at the Institute for Genome Sciences (University of Maryland - Baltimore). Read alignments to the V. cardui reference genome were generated using BWA-MEM. Local sequencing coverage was determined using Mosdepth. To normalise the data, coverage values for each window were divided by the average depth for the entire scaffold. Wild-type coverage was calculated by averaging the median normalised counts across 4 control V. cardui individuals, and used to compare to the G1 and G2 normalised counts, to account for any population specific structural variation.

本数据集源自红蛱蝶（Vanessa cardui）样本的重测序基因分型，其中包含通过双重靶向象牙长链非编码RNA（ivory lncRNA）外显子1与外显子2获得的基因编辑嵌合体（crispant）。研究人员将红蛱蝶的合胞体胚胎在产卵后45分钟至2小时（AEL）期间，通过注射靶向象牙长链非编码RNA第一或第二外显子的两种单向导RNA（sgRNAs）混合液完成编辑。将G0代中出现大片突变克隆的成年嵌合体个体以每笼4-15只的密度集中饲养并进行随机互交；随后将G1代个体再次以每笼4-15只的密度随机互交，以获得G2代个体。从表现出强烈[象牙]表型的红蛱蝶成虫胸部提取基因组DNA，构建双端150bp（PE 150）测序文库，并在马里兰大学巴尔的摩分校基因组科学研究所的Illumina NovaSeq6000 S4测序流动槽上完成测序。使用BWA-MEM工具将测序读段比对至红蛱蝶参考基因组；通过Mosdepth工具计算局部测序覆盖度。为实现数据标准化，将每个基因组窗口的覆盖度值除以对应基因组支架（scaffold）的整体平均测序深度。以4只野生型红蛱蝶个体的标准化覆盖度中位数的平均值作为野生型对照，用于与G1、G2代的标准化覆盖度进行比较，以消除群体特异性结构变异带来的影响。