Hiseq 4000 pe150

Genomic DNA was extracted using the OMEGA Bacterial DNA kit (Omega Bio-tek, Norcross, United) and was then sequenced on both the llumina HiSeq 4000-PE150 (Illumina, San Diego, CA, United States) and the PacBio RS II platforms (Pacific Biosciences, California, United States). We created a complete genome sequence for M. morganii L241 using Unicycler (Wick et al., 2017 (link)) by combining our llumina sequencing reads with PacBio sequencing reads. By using Unicycler (Wick et al., 2017 (link)), raw llumina reads were assembled using SPAdes, semi-global alignment was then performed by aligning PacBio reads to the assembly data, the llumina sequenceing reads were finally used to polish the genome assembly with Bowtie2 and Pilon. Additionally, online tools¹ were used to identify acquired antimicrobial resistance genes and replicon type of plasmid. This genome was annotated by the RAST server (Aziz et al., 2008 (link)), while the IS Finder database² was used to identify transposon and IS elements. The circular image of multiple plasmids comparisons was generated by the BLAST Ring Image Generator (BRIG) (Alikhan et al., 2011 (link)). Finally, the comparison figures of the genetic context of bla_NDM–5 on multiple plasmids were performed with a Python application Easyfig (Zheng et al., 2017 (link)).

DNA was extracted using MasterPure DNA extraction kit (Epicentre, USA) with proteinase K following the manufacturer’s instructions. DNA concentrations were measured using a NanoDrop One Spectrophotometer (Thermo Fisher Scientific, USA) and DNA extracts were stored at −20°C for metagenomic sequencing. Shotgun sequencing was performed using Illumina Hiseq 4000 PE150 (for sample SK) (Novogene, China) and Illumina Novaseq 6000 PE150 (for samples ST, STL, SWH, TP, and YL) (Novogene, China). Metagenomic sequencing for SK, ST, STL, SWH, TP, and YL yielded 40.1, 32.6, 34.1, 40.6, 33.8, and 41.7 Gb raw data, respectively. Read numbers for SK, ST, STL, SWH, TP, and YL were 267 million, 217 million, 227 million, 271 million, 225 million, and 278 million, respectively. The sequencing depth (average coverage for viral contigs) for SK, ST, STL, SWH, TP, and YL was 107×, 69×, 65×, 64×, 103×, and 139×, respectively.

Fecal sample processing, nucleic acid extraction, library preparation, and whole genome shotgun sequencing were performed at the University of North Carolina at Chapel Hill Microbiome Core (Chapel Hill, NC, USA), 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 2S Turbo DNA library kit. DNA was sequenced on the Illumina HiSeq 4000 PE 150 platform. Positive controls included ZymoBIOMICS Microbial Community Standard (Cat. No. D6300), Microbial Community DNA standard (Cat. No. D6305), and Gut Microbiome Standard (Cat. No. D6331). DNA-free deionized water was used as a negative control and to detect possible contamination. To avoid batch effects, fecal samples were randomized prior to nucleic acid extraction and all samples were sequenced at the same time. 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).

The total genomic DNA was extracted from 100 mg of fresh leaves by the modified CTAB method (Li et al., 2013 (link)). Subsequently, the total DNA was disrupted by ultrasound to produce fragments of 300–500 bp and the fragment quality was checked using Bioanalyzer 2100 (Agilent Technologies). A 400 bp DNA library was constructed using the NEB Next Ultra™ DNA Library Kit (Illumina, San Diego, California, USA). To assemble the chloroplast genome, we firstly using Hiseq 4000 PE 150 (Illumina, San Diego, California, USA) to sequencing the library fragments, then SPAdes (Bankevich et al., 2012 (link)) was used for de novo assemblies, the contigs obtained were further screened by BLAST, after that, Sequencher 4.10 (http://www.genecodes.com) were used to marge the screened contigs, finally, Geneious 8.1 (Kearse et al., 2012 (link)) was used to compare all reads to the spliced chloroplast genome sequence to test whether the counting sequence was correct or not.

At 7 days post-induction, total RNA was extracted. Polyadenylated RNA-seq was performed using standard Illumina Truseq kits, and samples were sequenced using the Illumina HiSeq 4000 PE150.

Genomic DNA were isolated from promastigotes representing each of the HCZ isolate, DD8 and 9044 strains of L. donovani using TIANamp Genomic DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. In brief, purified stationary phase promastigotes (~ 4 × 10⁸ cells) were transferred to a sterile microcentrifuge tube containing lysis buffer, from which the released genomic DNA was bound to column, washed with washing buffer, and then eluted with nuclease-free water. DNA samples representing the HCZ isolate, DD8 and 9044 strains were stored at -20 °C until use.
The quality and intensity of DNA samples were measured using a Qubit Fluorometer (Thermo Fisher Scientific, Rochester, NY, USA). Sequencing libraries (pair-end) representing each L. donovani samples (HCZ isoalte, DD8 and 9044 strains) were constructed using an internal DNA 200 bp-800 bp Insert Size Pooling Library Construction Kit (BGI, Shengzheng, China). In brief, 1 µg genomic DNA was randomly fragmented, incubated with End Repair Mix, combined with A-Tailing Mix, ligated to Illumina adapters, amplified with PCR Primer Cocktail and PCR Master Mix, and validated by the Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System. The qualified libraries were sequenced on Illumina Hiseq4000 (PE150) platform.