ONT Sequencing of Genomic DNA from Muscle

For ONT sequencing, we extracted genomic DNA from muscle using the phenol-chloroform-isoamyl alcohol extraction, followed by isopropanol precipitation. The DNA was further purified with NucleoBond AXG 100 columns with the NucleoBond Buffer Set IV (Macherey-Nagel, Germany). ONT libraries were prepared with the Ligation Sequencing Kit (SQK-LSK109; Oxford Nanopore Technologies, UK) and were sequenced using multiple R9.4.1 flow cells on an ONT GridION platform. The raw .fast5 files were base-called by Guppy v4.0.11 with the high-accuracy mode and quality filtering. Reads that were shorter than 1 kb were discarded using SeqKit v0.12.0 (Shen et al. 2016 (link)). The length-filtered ONT long reads were used in the downstream analyses. Supplementary Table S3 summarizes the filtered genomic reads generated in this study.

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Kawato S., Nishitsuji K., Arimoto A., Hisata K., Kawamitsu M., Nozaki R., Kondo H., Shinzato C., Ohira T., Satoh N., Shoguchi E, & Hirono I. (2021). Genome and transcriptome assemblies of the kuruma shrimp, Marsupenaeus japonicus. G3: Genes|Genomes|Genetics, 11(11), jkab268.

Publication 2021

NucleoBond AXG 100 columns NucleoBond Buffer Set IV Ligation Sequencing Kit (SQK-LSK109;

Buffer Cells Chloroform Genomic Guppy Isoamyl alcohol Isopropanol Ligation Muscle Phenol

Corresponding Organization : Okinawa Institute of Science and Technology Graduate University

Other organizations : The University of Tokyo, Kanagawa University

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Variable analysis

independent variables

DNA extraction method (phenol-chloroform-isoamyl alcohol extraction, followed by isopropanol precipitation)
DNA purification method (NucleoBond AXG 100 columns with the NucleoBond Buffer Set IV)
Library preparation method (Ligation Sequencing Kit SQK-LSK109)
Sequencing platform (ONT GridION)
Base-calling software (Guppy v4.0.11 with high-accuracy mode and quality filtering)
Read filtering (discarding reads shorter than 1 kb using SeqKit v0.12.0)

dependent variables

Filtered genomic reads generated in this study (summarized in Supplementary Table S3)

control variables

Not explicitly mentioned

controls

No positive or negative controls were explicitly mentioned in the provided information.

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