Dna sequencing reagent kit 4.0 v2

PacBio libraries were prepared using the SMRTbell Template Prep Kit 1.0/SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences). To prepare the RS II library, genomic DNA was sheared to 20 kbp using g-TUBE as performed for ONT libraries (Covaris) followed by DNA-damage repair and end-repair using polishing enzymes. Blunt-end ligation was used to create the SMRTbell template. To prepare the Sequel II CCS/HiFi library, genomic DNA was sheared to 15 kbp using the Megaruptor 2 (Diagenode). Unsheared Sequel II CLR and sheared CCS/HiFi libraries were ligated with overhang adapters. Library fragments were size-selected using BluePippin. SMRTbell Polymerase Complex was created using DNA/Polymerase Binding Kit P6 v2 for RSII libraries, Sequel II Binding Kit 1.0 for Sequel II CLR libraries, and Sequel II Binding Kit 2.0 for Sequel II CCS/HiFi libraries (Pacific Biosciences). PacBio RS II libraries were sequenced using DNA Sequencing Reagent Kit 4.0 v2 and RS II SMRT Cells v3 (Pacific Biosciences), with 4 h movie length. Sequel II CLR libraries were sequenced using Sequel II Sequencing Plate 1.0 and SMRT Cells 8 M (Pacific Biosciences) with 15 h movie length. Sequel II CCS/HiFi libraries were sequenced using Sequel II Sequencing Plate 2.0 and SMRT Cells 8 M with 30 h movie length.

As per [10 (link)], (a) amplification of full length 16S ribosomal gene (1464 bp) from 50 ng/μl DNA from each CP sample using V1-V9 variable region primers; (b) barcoding of each amplified amplicon using paired 16-nt symmetric barcodes (https://github.com/PacificBiosciences/Bioinformatics-Training/wiki/Barcoding-with-SMRT Analysis-2.3) (Table 1); (c) multiplexing of barcoded-purified amplicons in a group of 5 libraries (Supplementary Table 2); and (d) Sequencing using DNA Sequencing Reagent Kit 4.0 v2 (Pacific Biosciences, USA) in a total of 24 SMRT cells (3 cells/library) using 6-hour movies data collection protocol were performed.

Genomic DNA from the clinical strains was isolated by the phenol-chloroform method as previously described [48 ]. Sequencing was performed using the MiSeq system and PacBio RS-II. Sequencing libraries for MiSeq were prepared using the Nextera XT DNA Library Preparation Kit according to the manufacturer’s protocol (Illumina, CA). Each DNA library with adapters was normalized to 4 nM, pooled, and sequenced by the MiSeq system with MiSeq Reagent Kit v3 (Illumina, CA). Sequencing libraries for PacBio were prepared using SMRTbell Template Prep Kit 1.0 according to the manufacturer’s protocol (Pacific Biosciences, CA). For each SMRTbell DNA library, size selection was performed using BluePippin. Each size selected SMRTbell DNA library was sequenced by the PacBio RS-II system with DNA Sequencing Reagent Kit 4.0 v 2 (Pacific Biosciences, CA). All genome sequences, including the strains registered by other researchers in NCBI, were annotated using dFAST to standardize the data prior to pan-genomic analysis [49 (link)].