2.3 Long DNA library construction and sequencing
Long fragments of extracted
high-quality DNA from fresh muscle were selected using a Blue Pippin
System (Sage Science, MA, USA), with a peak value of 20 kb. After the
short reads were reduced, the long fragments were used for nanopore
sequencing. The sequencing adapters were ligated to the ends of the long
fragments, according to the manufacturer’s instructions for the 1D
Ligation Sequencing Kit (SQK-LSK109, Nanopore, Oxford, UK). Finally, the
20 kb genomic DNA libraries were quantified using a Qubit 3.0
Fluorometer (Invitrogen, Camarillo, USA). Meanwhile, three 20 kb
libraries (three 1D prep) were prepared and sequenced on one flow cell
using the Nanopore PromethION DNA sequencer (Oxford Nanopore, Oxford,
UK), according to the manufacturer’s instructions.