Native barcoding expansion kits 1 12 and 13 24

Whole-genome multiplex PCR was performed for both sequencing primer pools (A and B) separately (separated tubes). After PCR, the amplified products were purified using 1× AMPure XP beads (Beckman Coulter), and their concentrations were quantified using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit on a Qubit 3.0 fluorimeter (Thermo Fisher). Then, the concentrations for each pool were normalized based on the previous quantification in a single tube. DNA library preparation was conducted for all samples which presented a DNA concentration of >1 ng/μL after the cleanup procedure. For the library preparation, the ligation sequencing kit (Oxford Nanopore Technologies) and native barcoding expansion kits 1-12 and 13-24 (Oxford Nanopore Technologies) were used, following the reaction conditions previously described in reference 30 (link). One barcode was used for each sample, in order to optimize and increase the number of samples per flow cell in the same sequencing run. A sequencing library was generated using the SQK-LSK109 ligation sequencing kit (Oxford Nanopore Technologies) and loaded onto a R9.4 flow cell (Oxford Nanopore Technologies). Sequencing was performed for 8 h on a MinION device, and the final consensus sequences were obtained using Genome Detective software (https://www.genomedetective.com/) (31 (link)).

Whole-genome multiplex PCR was performed for both sequencing primer pools (A and B) separately (separated tubes). After PCR, the amplified products were purified using 1× AMPure XP beads (Beckman Coulter), and their concentrations were quantified using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit on a Qubit 3.0 fluorimeter (Thermo Fisher). Then, the concentrations for each pool were normalized based on the previous quantification in a single tube. DNA library preparation was conducted for all samples which presented a DNA concentration of >1 ng/μL after the cleanup procedure. For the library preparation, the ligation sequencing kit (Oxford Nanopore Technologies) and native barcoding expansion kits 1-12 and 13-24 (Oxford Nanopore Technologies) were used, following the reaction conditions previously described in reference 30 (link). One barcode was used for each sample, in order to optimize and increase the number of samples per flow cell in the same sequencing run. A sequencing library was generated using the SQK-LSK109 ligation sequencing kit (Oxford Nanopore Technologies) and loaded onto a R9.4 flow cell (Oxford Nanopore Technologies). Sequencing was performed for 8 h on a MinION device, and the final consensus sequences were obtained using Genome Detective software (https://www.genomedetective.com/) (31 (link)).