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... [ view full abstract ]

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 that leverages DNA molecules up to and above 100 kb in size. The core our platform is a microfluidics system that delivers haplotype-level dilution of long input molecules into >1 million barcoded partitions. Libraries are constructed within the partitions to generate barcoded DNA fragments that are sequenced on standard illumina platforms. Due to the limiting amount of DNA in the system, reads can typically be associated to the molecule of origin, creating a novel data type referred to as ‘Linked-Reads’.

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 and chemical 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). These samples are optimal for generating Linked-Read datasets and enable de novo diploid genome assembly and reference-based analysis including phased variant calling, identification of large-scale rearrangements, and structural variant discovery.

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.