Optimized Sample Prep Methods for Long-Range Genomics and de novo Assembly

Traditional methods for isolating high molecular weight genomic DNA are time consuming, require a significant amount of delicate handling, and utilize non-standard and/or low-throughput laboratory techniques. We have developed a scalable platform for long-range genome sequencing and assembly, leveraging DNA molecules up to and above 100 kb in size. In order to match the overall throughput of our technology (>24 samples per day), we have optimized methods for routine isolation of DNA >200 kb from small amounts of biological samples including whole blood (200 ul), cell suspensions (1e6 cells), and fresh-frozen tissue (25 mg). We have methodically identified and removed steps that cause both physical (double strand breaks) and chemical (single strand nicks, abasic sites) damage to DNA molecules during the isolation procedure, and ensured that our protocols are centered around widely available reagents (e.g. QIAGEN magnetic beads). Protocols range from 70 minutes (blood) to 3 hours (tissue), and the resulting DNA samples generate diploid de novo assemblies with a typical N50 scaffold size of >10 Mb (can be lower due to genome structure) using standard short-read sequencing. Further, since this is a diploid assembly method, the homologous chromosomes are separated and haplotypes are accurately reconstructed at multi-megabase scale. We specifically demonstrate equivalence of our optimized methods against the “gold standard” method for high molecular weight DNA isolation - agarose gel plugs - by comparing multiple assemblies including bird, reptile, and mammalian genomes.