low-pass nanopore sequencing of the Caribbean spiny lobster Panulirus argus
Muscle tissue was extracted from a pereiopod and total genomic DNA (gDNA) was isolated using the QIAGEN DNeasy Blood and Tissue Kit (QIAGEN USA) following the manufacturer's protocol. A total of five extractions were pooled together to obtainhave enough high molecular weight gDNA for preparing a single native barcoded ligation library using a modified version of the one-pot ligation protocol for Oxford Nanopore libraries (https://protocols.io/ view/one-pot-ligation-protocol-for-oxford-nanopore-libr-k9acz2e.html). In brief, gDNA concentration was measured using the Qubit 4 Fluorometer (Thermo Fisher Scientific) and purified for a first time using Ampure XP clean-up beads (Beckman Coulter, IN, USA) following the manufacturer's protocol. Next, a total of 200 fmol purified gDNA was sheared (to ~8, 000 bp) using a g-TUBE (Covaris, MA, USA) following the manufacturer's protocol. Sheared gDNA was purified for a second time using Ampure XP clean-up beads and then repaired using NEBNext FFPE DNA Repair Mix (New England Biolabs, MA, USA) and end-prepared (poly-A adding) using NEBNext Ultra II End Repair/dA-Tailing Mix (New England Biolabs, MA, USA) following the manufacturer's protocol. Ligation of a native Oxford Nanopore barcode (NB10 from the EXP-NBD003 native barcoding kit, ONT, Oxford, UK) to the dA-tailed gDNA was conducted using NEBNext Ultra II Ligation Module (New England Biolabs, MA, USA) following the manufacturer's protocol. The resulting barcode -ligated library was pooled together with other libraries and BAM 1D sequencing adapters were ligated using NEBNext Ultra II Ligation Module (New England Biolabs, MA, USA) following the manufacturer's protocol. Lastly, the pooled libraries were purified for a last time using Ampure XP clean-up beads and multiplex-sequenced for 48 hr on a MinION portable device with a MinION R9 flow cell (FLOMIN106, R9.4 chemistry) (ONT, Oxford, England) controlled with the software MiniKnow. The raw signal (FAST5 files) was basecalled using the Albacore Sequencing Pipeline Software (ONT, Oxford, England) obtaining a total of 77, 520 sequencing reads in FASTQ format