A high-quality reference genome is an essential resource for primary and applied research across the tree of life. Genome projects for small-bodied, non-model organisms such as insects face several unique challenges including limited DNA input quantities, high heterozygosity, and difficulty of culturing or inbreeding in the lab. Recent progress in PacBio library preparation protocols, sequencing throughput, and read accuracy address these challenges. We present several case studies including the Red Admiral (Vanessa atalanta), Monarch Butterfly (Danaus plexippus), and Anopheles malaria mosquitoes that highlight the benefits of sequencing single individuals for de novo genome assembly projects, and the ease at which these projects can be conducted by individual research labs. Sampled individuals may originate from lab colonies of interest to the research community or be sourced from the wild to better capture natural variation in a focal population. Where genomic DNA quantities are limited, the PacBio Low DNA Input Protocol requires ~100 ng of input DNA. Low DNA input samples with 500 Mb genome size or less can be multiplexed on a single SMRT Cell 8M on the Sequel II System. For samples with more abundant DNA quantity, size-selected libraries may be constructed to maximize sequencing yield. Both low DNA input and size-selected libraries can be used to generate HiFi reads, whose quality is Q20 or above (1% error or less) and lengths range from 10 – 25 kb. With HiFi reads, de novo assembly computation is greatly simplified relative to long read methods due to smaller sequence file sizes and more rapid analysis, resulting in highly accurate, contiguous, complete, and haplotype-resolved assemblies.
June 1, 2021 |