Pacbio sequel sequencing platform

Long-read genome sequencing was performed on the PacBio Sequel sequencing platform by the Georgia Genomics and Bioinformatics Core facility at the University of Georgia. A large insert library was constructed for each strain using the SMRTbell Template Prep Kit following the PacBio’s instructions for >20 kb Template Preparation using BluePippin Size-Selection System for Sequel Systems. Fragment Analyzer analyses showed the final size of the insert libraries was 37 kb for NCYC 4144, and 22-24 kb for NCYC 4145 and NCYC 4146. Primers were annealed to the templates, then templates were bound to polymerase using the Sequel Binding Kit 2.0. The resultant polymerase bound complexes were purified using SMRTbell Clean Up Columns for Sequel, and were then bound to MagBeads for loading. Genomic DNA for each strain was loaded onto a single SMRTcell v1 and run on the PacBio Sequel System with a movie time of 10 hr.

The whole-genome shotgun strategy was adopted to construct the libraries of different inserted fragments, and next-generation sequencing was performed on an Illumina NovaSeq sequencing platform (San Diego, CA, USA). Third-generation single-molecule sequencing technology was used to sequence these libraries on the PacBio Sequel sequencing platform (Menlo Park, CA, USA). The analysis of antibiotic resistance was based on the Comprehensive Antibiotic Resistance Database (https://card.mcmaster.ca/; accessed on 1 July 2021) [44 (link)].

The total genomic DNA of each soil sample (200 mg) was extracted using the Fast DNA Spin Kit for soil (MP Biomedicals, Solon, OH, USA) according to the manufacturer’s instructions. The full length of the bacterial 16 S rRNA gene was amplified using the primer sets 27 F (5ʹ-AGRGTTTGATYNTGGCTCAG-3ʹ) and 1492R (5ʹ-TASGGHTACCTTGTTASGACTT-3ʹ), and followed thermal conditions: initial denaturation at 95 °C for 3 min, followed by 27 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s, and 72 °C for 10 min. The purified amplicons were sequenced using the single molecule real-time sequencing (SMRT) method and a PacBio Sequel sequencing platform (PacBio Menlo Park, CA, USA) according to the standard protocol. The aforementioned operations were completed by Biomarker Technologies Corporation (Beijing, China).

The whole genome of Nguyenibacter sp. L1 was sequenced using the Illumina NovaSeq sequencing platform (Illumina, San Diego, CA). A Whole Genome Shotgun (WGS) strategy was adopted to construct various insert libraries, which were then sequenced using both next-generation sequencing (NGS) on the Illumina HiSeq sequencing platform and third-generation single-molecule sequencing technology on the PacBio Sequel sequencing platform. Subsequent assembly of the third-generation sequencing data was carried out using HGAP3 (Chen et al., 2016 (link)) and CANU1.8 (Koren et al., 2017 (link)). Pilon1.23 was then employed to apply corrections from the high-quality next-generation data to the third-generation contig results (Walker et al., 2014 (link)), thereby yielding the complete sequence. The complete procedures for whole-genome sequencing and assembly were executed by Personal Gene Technology Co., Ltd. (Nanjing, China). The sequencing data pertinent to this publication has been deposited in the NCBI’s short read archive under the accession number PRJNA975354.