Location: Stored Product Insect and Engineering Research
Title: Optimized extraction of insect genomic DNA for long-read sequencingAuthor
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Oppert, Brenda |
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Stoss, Samantha |
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Monk, Alaysha |
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Smith, Timothy |
Submitted to: Methods and Protocols
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 11/18/2019 Publication Date: 11/23/2019 Citation: Oppert, B.S., Stoss, S.P., Monk, A.L., Smith, T.P. 2019. Optimized extraction of insect genomic DNA for long-read sequencing. Methods and Protocols. 2(4):89. https://doi.org/10.3390/mps2040089. DOI: https://doi.org/10.3390/mps2040089 Interpretive Summary: As DNA sequencing technologies continue to improve the lengths of DNA that can be read has increased, the challenge in the laboratory is to extract DNA of sufficient fragment size and quality to support the longer read lengths. We developed and optimized a procedure for obtaining high quality long genomic DNA from insects. We first determined that the optimal developmental stage of insects for genomic DNA extraction was the pupal stage because it avoids complications by the presence of DNA from ingested food and other types of contamination. Improved results were obtained by gently disrupting soft pupal tissue in initial buffers using Teflon micropestles, rather than grinding frozen tissue. We demonstrate a modified procedure using a commercial DNA extraction kit, with pooled pupae of red flour beetle. Other modifications included avoiding vortexing steps, instead gently mixing by inversion of the tube, and using wide bore pipettes while transferring fractions containing genomic DNA. One sample was selected for long read sequencing, and we provide evidence of successful downstream library production and sequencing of the sample. While the technique has been optimized for insects, extractions from tissues of other organisms using these modified procedures may also support successful long read sequencing. Technical Abstract: Long read sequencing technologies have continued to increase the length of reads that are possible, and at present can average read lengths of >20 kb up to as much as 60-80 kb. The challenge in the laboratory side of genome assembly has therefore moved from read length limitations of the technologies, to extracting DNA of sufficient fragment size and quality to support the read lengths already achievable. We developed and optimized a procedure for obtaining high quality long genomic DNA from insects. We first determined that the optimal developmental stage of insects for genomic DNA extraction was the pupal stage because it avoids complications by the presence of DNA from ingested food, and reduces contamination with chitinous material that can interfere with extraction and downstream sequencing processes. Improved results were obtained by gently disrupting soft pupal tissue in initial buffers using Teflon micropestles, rather than grinding frozen tissue. We demonstrate a modified procedure using a commercial DNA extraction kit, with pooled pupae of red flour beetle, Tribolium castaneum. Modifications included avoiding vortexing steps, instead gently mixing by inversion of the tube, and using wide bore pipettes while transferring fractions containing genomic DNA. One sample was selected for long read sequencing, and we provide evidence of successful downstream library production and sequencing of the sample. While the technique has been optimized for insects, extractions from tissues of other organisms using these modified procedures may also support successful long read sequencing. |