Abstract
Long-read sequencing technologies continue to increase the length of reads, and at present can average read lengths of >20 kb up to 60–80 kb. Now the challenge is to extract genomic DNA of sufficient fragment size and quality to support longer read lengths. We developed a successful method to consistently obtain high-quality long genomic DNA from insects. The optimal developmental stage of insects for genomic DNA extraction was determined to be the pupal stage, eliminating DNA from ingested food and reducing contamination by chitinous material that can interfere with extraction. Improved results were obtained by a modified procedure of a commercial genomic DNA extraction kit. Initially, soft pupal tissue of the red flour beetle, Tribolium castaneum, was disrupted in the kit lysis buffer using Teflon micropestles. Modifications to the kit protocol also included gentle mixing by inversion of the tube, instead of harsh vortexing steps, and using wide-bore pipette tips in transferring fractions containing genomic DNA. Data from one sample were provided as an example of successful downstream library production and sequencing. While the technique has been optimized for insects, extractions from tissues of other organisms using these modified procedures also may improve long-read sequencing results.
Highlights
Long-read sequencing is increasingly being used for a number of biological applications, especially genome assembly
We evaluated a number of commercial kits for improvements to genomic DNA extractions from stored product insects for long-read sequencing
We found one kit superior in the reproducible extraction of high-quality long genomic DNA, and we demonstrate our modified procedure with a common stored product pest, Tribolium castaneum
Summary
Long-read sequencing is increasingly being used for a number of biological applications, especially genome assembly. A method for the isolation of eukaryotic high-molecular-weight DNA was proposed by Blin and Stafford [2], involving homogenization of tissues in liquid nitrogen in a Waring Blender, and achieving nick-free DNA of 200 × 106 Da (about 300,000 bp) Another method for low cost and rapid genome. All kits had the advantage of not generating hazardous waste of phenol and chloroform, but the DNeasy kit offered the shortest extraction time, and the Puregene kit had lowest contamination of protein These researchers found that using up to 8× volumes of ethanol and 4 ◦C enhanced the amount of DNA extracted, the increased volume of ethanol increased the cost of extraction, and lower temperatures tended to make the DNA solution more viscous with the potential to clog columns. We found one kit superior in the reproducible extraction of high-quality long genomic DNA, and we demonstrate our modified procedure with a common stored product pest, Tribolium castaneum (the red flour beetle)
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