| Popis: |
The requirement for large amounts of purified DNA limits many sequencing experiments, especially when seeking to avoid pre-amplification or when using third generation technology to sequence molecules directly. We wanted to test the limits of current nanopore sequencing input requirements and devised a set of experiments to evaluate extraction and library preparation approaches for low inputs.We found an optimised bead beating approach combined with a magnetic bead protocol, rather than traditional spin columns for DNA extraction, improved both molecule length, integrity score and DNA yield. Through reducing the DNA input to as little as 6.25 % of recommended (25 ng versus 400 ng) and reaction volumes in half, library construction can be completed, and sequencing begun within 20 minutes of sample collection.Applying these approaches, we demonstrated that our pipeline can be used as a cheap and effective method to de novo assemble a genome and identify genes from low quantities and quality of DNA. With our rapid extraction protocol using transportable equipment and low input library construction we were able to generate a de novo assembly from a single insect (Drosophila melanogaster) spanning 125 Mbp / 85 % of the reference genome, over 96.9% complete BUSCO genes, with a contig N50 over 1.2 Mbp, including chromosome arm sized contigs, for a modest consumable cost under £600. |
