Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform
Pacharaporn Angthong,
Tanaporn Uengwetwanit,
Wirulda Pootakham,
Kanchana Sittikankaew,
Chutima Sonthirod,
Duangjai Sangsrakru,
Thippawan Yoocha,
Intawat Nookaew,
Thidathip Wongsurawat,
Piroon Jenjaroenpun,
Wanilada Rungrassamee,
Nitsara Karoonuthaisiri
Affiliations
Pacharaporn Angthong
Microarray Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand
Tanaporn Uengwetwanit
Microarray Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand
Wirulda Pootakham
National Omics Center, National Science and Technology Development Agency, Pathum Thani, Thailand
Kanchana Sittikankaew
Microarray Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand
Chutima Sonthirod
National Omics Center, National Science and Technology Development Agency, Pathum Thani, Thailand
Duangjai Sangsrakru
National Omics Center, National Science and Technology Development Agency, Pathum Thani, Thailand
Thippawan Yoocha
National Omics Center, National Science and Technology Development Agency, Pathum Thani, Thailand
Intawat Nookaew
Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States of America
Thidathip Wongsurawat
Division of Bioinformatics and Data Management for Research, Department of Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
Piroon Jenjaroenpun
Division of Bioinformatics and Data Management for Research, Department of Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
Wanilada Rungrassamee
Microarray Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand
Nitsara Karoonuthaisiri
Microarray Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand
Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.
