Optimization of capture protocols across species targeting up to 32000 genes and their extension to pooled DNA

In-solution based capture is becoming a method of choice for sequencing targeted sequence. We assessed and optimized a capture protocol in 20 different species from 6 different plant genus using kits from 20,000 to 200,000 baits targeting from 300 to 32,000 genes. We evaluated both the effectiveness of the capture protocol and the fold enrichment in targeted sequences. We proposed a protocol with multiplexing up to 96 samples in a single hybridization and showed it was an efficient and cost-effective strategy. We also extended the use of capture to pools of 100 samples and proved the efficiency of the method to assess allele frequency. Using a set of various organisms with different genome sizes, we demonstrated a correlation between the percentage of on-target reads vs. the relative size of the targeted sequences. Altogether, we proposed methods, strategies, cost-efficient protocols and statistics to better evaluate and more effectively use hybridization capture. ### Competing Interest Statement The authors have declared no competing interest.