Pacbio rs 2 platform

Six long-read datasets from four yeast sample strains (Kagoshima no.2, BY4742, SY14 and NRRL Y-567) were collected from NCBI database (Table S1). The corresponding sample IDs were SAMD00082707, SAMN08364553, SAMN08364554, and SAMN09475318, respectively. SAMD00082707 was Kagoshima no. 2 strain from PRJDB5836 and used to brew the Japanese distilled spirit shochu. The long read of SAMD00082707 was sequenced 252-fold by three cells run on the PacBio RSII platform (Mori et al., 2017 (link)). The samples of SAMN08364553 and SAMN08364554 were wild-type strain BY4742 and SY14 from PRJNA429985 (Shao et al., 2018 (link)), which were sequenced by PacBio Sequel with 317-fold and 411-fold coverage, respectively. The sample SAMN09475318 from PRJNA477598 was the NRRL Y-567 strain, which was sequenced and generated three datasets in three instruments including PacBio Sequel, PacBio RSII, and ONT GridION (1D ligation kit and R9.5 flow cell) (Mcintyre et al., 2019 (link)). The collected six SRA format files were converted to FASTQ by using the NCBI SRA Toolkit (version 2.9.4).

Total genomic DNA of FBFS97 was extracted using the GenElute Bacterial Genomic DNA kit (Sigma) according to the manufacturer’s recommendations. FBFS97 whole genome was sequenced using the PacBio RSII platform utilizing 2 single- molecule real-time (SMRT) cells, was used to acquire the raw sequence reads at the Genome Technology Facility (GTF) in Lausanne, Switzerland. The high- quality reads were then assembled de novo into a single contig using Hierarchical Genome Assembly Process version 3.0 (HGAP 3.0) in SMRT Analysis version 2.3.0. Automatic annotation and gene prediction were carried out using Prokka version 1.1.0. Circos was used to create the genome circle (https://circos.ca/tutorials/lessons/).

The genomic DNA of strain SP-35 was extracted using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI) and whole-genome sequencing was performed by the Majorbio Company (Shanghai) using the PacBio RS II platform and done using the single molecule real time (SMRT) sequencing. A SMRT bell sequencing library with a 7–8 kb insert size was prepared using the PacBio SMRTbell Template Prep Kit 1.0. Afterwards, the raw data was assembled with the assistance of a Celera assembler and polished by Quiver tool (version 1.1.0) [15 (link)].

Total RNA was isolated using a UNIQ-10 column TRIzol total RNA extraction kit (Sangon Biotech) according to the manufacturer’s instructions, followed by treatment with DNase I. The mRNA was purified by a poly T column separation and stored at −80 °C until further analysis. The Iso-Seq library was prepared according to the PacBio Isoform Sequencing protocol (Iso-Seq™). The RNA was reverse transcribed using a SMARTer® PCR cDNA Synthesis Kit and was PCR amplified using KAPA HiFi PCR Kits. These cDNA products were purified using a SMRTbell DNA Template Prep Kit 3.0 for library construction. The libraries were sequenced using P6C4 polymerase and chemistry on a PacBio RS II platform with 240 min movie times at Tianjin Lakeside Powergene Science Development Co. Ltd. In total, 7 SMRT Cells were used to generate 4.4 Gbp of transcriptome cDNA sequencing data.

Genomic DNA was isolated from frozen fecal samples using a DNA isolation kit (MoBio, Carlsbad, CA, United States) and quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). 16S rRNA genes (V3-V4 region) were PCR amplified using the forward primer 341F (5′-ACTCCTACGGGRSGCAGCAG-3′) and reverse primer 806R (5′-GGACTACVVGGGTATCTAATC-3′), purified using Agencourt AMPure magnetic purification beads (Beckman Coulter, Brea, CA, United States), and run on 2% agarose gel. High-throughput sequencing of the PCR products was carried out on the PacBio RS II platform and analyzed at Ruiyi Biotechnology Co., Ltd. (Hangzhou, China).
Raw data from high-throughput sequencing were demultiplexed and quality filtered using the QIIME2 platform, then used to assemble operational taxonomic units (OTUs) to define species, genus, or class of bacterial communities by UCLUST algorithm (an exceptionally fast sequence clustering program for nucleotide and protein sequences) with a threshold of 97%. Principal component analysis (PCA) plots were generated using R software (v 4.1.2). STAMP (Ver. 2.1.3) software was applied to identify intestinal microbial phylotypes.

We previously sequenced and submitted annotated genomes of NTHi strains 10P129H1 (modA15) and 84P36H1 (modA18)²³ . Briefly, DNA was sequenced at the Yale Center for Genome Analysis (YCGA) using the PacBio RS II platform with P6-C4 chemistry and a library size of 10 kb, with one strain per SMRT cell, and assembled de novo using the hierarchical genome assembly process (HGAP)^{26 (link)}. Polishing for a pure-PacBio assembly was carried out using the Quiver algorithm^{26 (link)} from the SMRT Analysis software suite (version 2.3.0 – http://www.pacb.com/devnet/) with default parameters. Consensus sequences were submitted to NCBI for annotation with the Prokaryotic Genome Annotation Pipeline (PGAP), and annotated sequences submitted to GenBank (accession numbers CP029620 [strain 10P129H1] and CP029621 [strain 84P36H1]). Methylome analysis was carried out as described previously^{27 (link),28 (link)} by YCGA. Each genome was sequenced to sufficient depth to allow methylome analysis of m6A methylation (cytosine methylation was not searched for). Coverage of each strain was as follows - strain 10P129H1 - ModA15 ON coverage of 393.5 fold, and ModA15 OFF coverage of 435.5 fold; strain 84P36H1 – ModA18 ON coverage of 435.5 fold, ModA18 OFF coverage of 380.0 fold.

A single colony of K. flava S43 was inoculated to a 50-ml centrifuge tube with 10 ml of GYM4 artificial seawater (26.52 g NaCl, 5.228 g MgCl₂6H₂O, 3.305 g MgSO₄, 1.141 g CaCl₂, 0.725 g KCl, 0.202 g NaHCO₃, 0.083 g NaBr, and 1 L distilled water) medium (10 g glucose, 4 g yeast extract, 4 g malt extract, and 1 L artificial seawater) (Schneemann et al., 2010 (link)). After incubation at 28°C and 180 rpm for 2 days, the culture was collected by centrifugation and its genomic DNA was extracted using a DNeasy^® Blood and Tissue Kit (Qiagen, United States). The integrity, concentration and purity of genomic DNA were detected by Agarose Gel Electrophoresis, Nanodrop and Qubit Fluorimeter. The genome was sequenced by the third generation sequencing (TGS), i.e., the Pacific Biosciences (PacBio) Single Molecule Real-Time (SMRT) sequencing technology. A whole-genome sequencing library was constructed using the SMRTcell Template Prep Kit (PacBio, United States) according to the manufacturer’s protocol, and the library was sequenced by using g-Tube on the PacBio RS II platform (PacBio).