Meeting Abstract
While using methods of genomic-DNA targeted gene capture to build a phylogeny of chitons, we encountered challenges within the protocols. Gene capture methods aim to sequence highly conserved gene regions of interest from genomic libraries using oligo probes and high throughput sequencing. The method is just now becoming more commonly used in non-model organisms. Although gene capture promises an efficient protocol for massively paralleled sequencing, we here address several hurdles and how to overcome them. First, small amounts of DNA are a challenge. Therefore, optimizing genomic DNA extractions is important to ensure that there is an abundance of gene fragments of interest, which should increase the probability of gene capture. Second, the presence of adapter-dimers—a common artifact from Illumina genomic library preparation—complicates the gene capture because the probes can non-specifically bind to them. Quality control via Bioanalyzer or TapeStation can reveal the adapter-dimers and size selection of DNA fragments can remove adapter-dimers. A third hurdle involves the optimization of the hybridization temperature between probe and gene target. We find it is important to heat denature the gene fragments at 98°C and slowly cool down to 65°C to enable the hybridization of single stranded gene targets and the probe. Lastly, figuring out how much sequencing coverage is needed per sample can be an ambiguous hurdle. A gene capture does not necessitate as many sequencing reads as a single transcriptome for example. One can calculate how much coverage one needs according to the number and length of the conserved genes of interest that the probes were designed from. It is necessary to discuss these challenges to minimize time and monetary costs while troubleshooting the multifaceted gene capture protocol for non-model organisms.
