Optimized Extraction of Insect Genomic DNA for Long-Read Sequencing
1
USDA Agricultural Research Service Center for Grain and Animal Health Research, 1515 College Ave., Manhattan, KS 66502, USA
2
USDA Agricultural Research Service U.S. Meat Animal Research Center, Clay Center, NE 67432, USA
*
Author to whom correspondence should be addressed.
Methods Protoc. 2019, 2(4), 89; https://doi.org/10.3390/mps2040089
Received: 24 October 2019 / Revised: 15 November 2019 / Accepted: 19 November 2019 / Published: 23 November 2019
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.
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Keywords:
genomic DNA; long-read sequencing; Oxford Nanopore; PacBio; red flour beetle; Tribolium castaneum
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MDPI and ACS Style
Oppert, B.; Stoss, S.; Monk, A.; Smith, T. Optimized Extraction of Insect Genomic DNA for Long-Read Sequencing. Methods Protoc. 2019, 2, 89. https://doi.org/10.3390/mps2040089
AMA Style
Oppert B, Stoss S, Monk A, Smith T. Optimized Extraction of Insect Genomic DNA for Long-Read Sequencing. Methods and Protocols. 2019; 2(4):89. https://doi.org/10.3390/mps2040089
Chicago/Turabian StyleOppert, Brenda; Stoss, Samantha; Monk, Alaysha; Smith, Timothy. 2019. "Optimized Extraction of Insect Genomic DNA for Long-Read Sequencing" Methods Protoc. 2, no. 4: 89. https://doi.org/10.3390/mps2040089
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