Hiseq 4000 pe150 platform

Genomic DNA of both NDM-5 producing C. freundii isolates was extracted using the QIAmp DNA Mini Kit (Qiagen, Germany). A Qubit Fluorometer (Thermo scientific, USA) was then used to determine the concentration and purity of DNA. Sequencing libraries were prepared using the Illumina Nextera XT Kit and sequenced using Illumina HiSeq 4000-PE150 platform (Illumina, USA). Raw sequencing data of both isolates were assembled using SOAP de novo software [34 (link)] and were deposited in GenBank under the following accession numbers: JAJDSQ000000000, and CP086287-CP086290, respectively. Antimicrobial resistance genes and plasmid replicon types were matched to the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/) Resfinder and Plasmid finder databases. The gaps were covered using combinatorial PCR to accomplish sequence integrality of contigs. The RAST server (http://rast.nmpdr.org/) was used to annotate the bacterial genomes, and the ISFinder database (https://www-is.biotoul.fr/) was used to identify IS elements and transposons. Multiple plasmid alignment was conducted and plotted between the bla_NDM-5-harboring plasmid (named pNDM-5) and the reference plasmid using the BLAST Ring Image Generator (BRIG) [35 (link)]. Easyfig 2.2.3 was used to analyze the genetic environment surrounding the bla_NDM-5 resistance gene [36 (link)].

Genomic DNA was extracted from fresh young leaves of the F. suspensa plant using the mCTAB method [47 ]. Genomic DNA was fragmented into 400–600 bp using a Covaris M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). Library preparation was conducted using NEBNext^® Ultra™ DNA Library Prep Kit Illumina (New England, Biolabs, Ipswich, MA, USA). Sample sequencing was carried out on an Illumina Hiseq 4000 PE150 platform.
Next, raw sequence reads were assembled into contigs using SPAdes [48 (link)], CLC Genomics Workbench 8 (Available online: http://www.clcbio.com), and SOAPdenovo2 [49 (link)], respectively. Chloroplast genome contigs were selected by BLAST (Available online: http://blast.ncbi.nlm.nih.gov/) [50 (link)] and were assembled by Sequencher 4.10 (Available online: http://genecodes.com/). All reads were mapped to the cp genome using Geneious 8.1 [51 (link)], which verified the selected contigs. The closing of gaps was accomplished by special primer designs, PCR amplification, and Sanger sequencing. Finally, we obtained a high-quality complete F. suspensa cp genome, and the result was submitted to NCBI (Accession Number: MF579702).

Cetyltrimethylammonium bromide (CTAB) method was used to extract genomic DNA from the LR mutant 3–15. Genome sequencing was conducted on the Illumina HiSeq 4000 PE150 platform using 150 bp paired-end libraries with 500 bp inserts at Wuhan SeqHealth Technology Company. We used Lofreq (version 2.1.5) software to perform SNP and InDel assays. In order to reduce unnecessary mutation sites, we specified strict screening conditions: 1. The mutation appears in the open reading frame region; 2. The mutation can only appear once in a gene; 3. The candidate gene is expressed in the hyphal growth stage; 4. The candidate gene is not mutated in the 1a_mut genome because the colony morphology of 3–15 is completely different from that of 1a_mutant, a mutant with moderate resistance to IPT.

The resistant mutant 1a_mut, MoIRR knockout transformant ΔMoIRR-1, and parental isolate H08-1a were inoculated into 100 ml flasks containing 40 ml PDB and shaking culture at 27°C, 150 rpm for 72 h. To ensure reliable gene expression profiles, each isolate had three biological replicates. Total RNA was extracted from the mycelia using Trizol reagent (Invitrogen, Carlsbad, CA, United States). Nine RNA samples were sent to Novogene Corporation (Novogene, Beijing, China) for RNA sequencing and basic analysis. RNA-Seq was performed on an Illumina HiSeq 4000 PE150 platform using 150 bp paired-end libraries with a 500-bp insertion. DEGs were calculated based on the normalized read count, and their corresponding values of p were determined. Pearson’s correlation coefficient was used to measure the expression profile similarities between samples. Differentially expressed genes were analyzed using the KEGG pathway database and enriched into KEGG pathways (Kanehisa et al., 2008 (link)). RNA-Seq reads data of H08-1a, ΔMoIRR-1, and 1a_mut were deposited at the GenBank SRA database under accession numbers SRX3362774, SRX3362767, SRX3362768, SRX3362771, SRX3362772, SRX3362773, SRX3362769, SRX3362770, and SRX3362775.

Fecal sample processing, nucleic acid extraction, library preparation, and sequencing were performed at the University of North Carolina at Chapel Hill Microbiome Core, which is supported by the following grants: Gastrointestinal Biology and Disease (CGIBD P30 DK034987) and the UNC Nutrition Obesity Research Center (NORC P30 DK056350. DNA was extracted using the QIAamp Fast DNA Stool Mini Kit and library was prepared using the Swift 2 S Turbo DNA library kit. RNA was extracted using the Qiagen RNeasy PowerMicrobiome kit and library was prepared using QIAseq Stranded Total RNA Library kit. DNA and RNA were sequenced on the Illumina HiSeq 4000 PE 150 platform. Mean total reads were 18,339,758, with similar read depth on each diet (19,475,004 for the WD and 17,204,513 for the MBD).