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