Microbial DNA in colonic chyme was obtained using the E.Z.N.A.® soil DNA kit (Omega Bio-Tek, Norcross, GA, United States), according to the builder's standard method. The V3-V4 hypervariable regions of the microbial 16S rRNA gene were amplified with PCR using primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) via the ABI Gene Amp® 9700 PCR thermocycler (ABI, CA, United States). Metagenomic sequencing was executed according to the Illumina platform by Miseq PE300. Raw reads were deposited into the NCBI Sequence Read Archive database (SRA: PRJNA765910). Raw sequences were treated and filtered into chimeric sequences by the Majorbio I-Sanger Cloud Platform (www.i-sanger.com , Majorbio, Shanghai, China). The Majorbio I-Sanger Cloud Platform (www.i-sanger.com , Majorbio, Shanghai, China) was used to perform unweighted principal coordinate analysis (PCoA), beta-diversity analysis, and alpha-diversity analysis using default values.
I sanger cloud platform
Manufactured by Majorbio
Sourced in China
The I-Sanger Cloud Platform is a lab equipment product that provides a centralized data management and analysis solution for DNA sequencing projects. It enables secure storage, processing, and sharing of sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Truseq rna sample preparation kit, by Illumina (4 mentions)
Miseq platform, by Illumina (3 mentions)
Miseq pe300 platform, by Illumina (2 mentions)
Qiaamp dna stool mini kit, by Qiagen (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Prism 8, by GraphPad (2 mentions)
Prism 7, by GraphPad (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Miseq pe300, by Illumina (2 mentions)
E z n a soil dna kit, by Omega Bio-Tek (2 mentions)
Sas 8, by SAS Institute (2 mentions)
Rna seq, by Majorbio (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Spss statistics v23, by IBM (1 mentions)
Omega soil dna kit, by Omega Bio-Tek (1 mentions)
Fecal dna isolation kit, by Qiagen (1 mentions)
Miseq pe250, by Illumina (1 mentions)
Powersoil dna isolation kit, by Qiagen (1 mentions)
Bioanalyzer 2100 expert, by Agilent Technologies (1 mentions)
60 protocols using i sanger cloud platform
1
Microbial DNA Extraction and 16S Sequencing
2
Microbial Community Analysis Protocol
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The free Majorbio I-Sanger Cloud Platform was used to conduct HTS analysis (www.I-Sanger.com , accessed on 16 March 2020). Both QIIME software (version 1.9.1; available at http://qiime.org , accessed on 16 March 2020) and Arch software (version 7.0; available at http://www.drive5.com , accessed on 16 March 2020) were used, as well as the Mothur program (version 1.30.2, available at mothur.org, accessed on 16 March 2020). The Observed, Chao1, and ACE indices were used to measure community diversity, while the Shannon and Simpson indices were used to measure community richness. PICRUSt (version 1.1.0, http://PICRUSt.github.io , accessed on 16 March 2020) was used to forecast the distribution of homologous gene clusters (COGs) based on 16S rRNA gene sequences.
To assess their differences, an analysis of alpha diversity was conducted using the Wilcoxon test. Based on Bray–Curtis phase dissimilarity, beta diversity was evaluated and visualized using principal coordinate analysis (PCoA). To evaluate the differences between the two groups, an analysis of similarity (ANOSIM) was conducted. R was used to conduct the statistical analysis and mapping of PCoA and ANOSIM. Additionally, linear discriminant analysis (LDA) was used to perform biomarker analysis of effect sizes (LEfSe) in order to pinpoint the bacterial taxa most likely to result in taxon demarcation.
To assess their differences, an analysis of alpha diversity was conducted using the Wilcoxon test. Based on Bray–Curtis phase dissimilarity, beta diversity was evaluated and visualized using principal coordinate analysis (PCoA). To evaluate the differences between the two groups, an analysis of similarity (ANOSIM) was conducted. R was used to conduct the statistical analysis and mapping of PCoA and ANOSIM. Additionally, linear discriminant analysis (LDA) was used to perform biomarker analysis of effect sizes (LEfSe) in order to pinpoint the bacterial taxa most likely to result in taxon demarcation.
3
Comprehensive Bioinformatics Analysis of Microbiome
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The data generated from PacBio and Illumina platform were used for bioinformatics analysis. All of the analyses were performed using I-Sanger Cloud Platform (www.i-sanger.com ) from Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). The coding sequence (CDS) was predicted with Glimmer v3.02. The tRNA and rRNA were predicted with tRNA-scan-SE v2.0 and Barrnap v0.8. The prophages was predicted using PHAge Search Tool. The gene island (GI) was predicted with Islander v1.2. The predicted CDSs were annotated from the NCBI non-redundant (NR) database, the databases of Swiss-Prot, The Pfam database, Cluster of Orthologous Groups of proteins (COGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGGs), Carbohydrate-Active enzymes (CAZy), Comprehensive Antibiotic Resistance Database (CARD) and Pathogen Host Interactions (PHI) using sequence alignment tools such as BLAST v2.3.0+, Diamond v0.8.35 and HMMER v3.1b2 [52 (link)].
4
T24 Cell Transcriptome Profiling
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After transfection with the vector or RhoC-Q63E, total RNA of T24 cells was extracted using the TRIzol reagent. The RNA sequencing (RNA-Seq) was performed by Majorbio (Shanghai, China). The data were analyzed on the free online platform of Majorbio I-Sanger Cloud Platform (www.isanger.com). The RNA-Seq data are available under GEO accession GSE129295.
5
Multivariate Analysis of Plasma Metabolites
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The multivariate statistical analysis for plasma metabolites was conducted using the ropls package (version 1.6.2) in R on the Majorbio I-Sanger Cloud Platform. The positive and negative data were obtained and analyzed using the SIMCA-236 P software package. After the metabolite variables were scaled to unit-variances, principal component analysis (PCA) was performed to visualize the metabolic differences among experimental groups using an unsupervised method. After the metabolite variables were scaled to Pareto scaling, orthogonal PLS-DA (OPLS-DA) was applied for statistical analysis to visualize the global metabolic alterations among comparable groups. To avoid the risk of overfitting, the model parameters R2 and Q2 were computed to evaluate the interpretability and predictability of the models. Variable importance in the projection (VIP) was calculated in the OPLS-DA model to present the overall contribution of each variable. If the VIP > 1.0, the variables are considered relevant for group discrimination. The p-values were estimated on one-dimensional statistical analysis with the paired Student’s t-test, and differences were declared at p < 0.05.
6
Statistical Analysis of Sequencing Data
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The statistical data were analyzed by the procedure of SAS (version 9.3, SAS Institute Inc., Cary, NC, USA). Duncan's multiple range tests were used to evaluate differences among treatments. High-throughput sequencing data were performed using an online platform of Majorbio I-Sanger Cloud Platform (www.i-sanger.com ).
7
Illumina-based Transcriptome Analysis Using I-Sanger Cloud
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The data generated from the Illumina platform were used for bioinformatics analysis. All the analyses were carried out using the I-Sanger cloud platform (www.i-sanger.com ) from Majorbio Bio-Pharm Technology Co., Ltd (Shanghai, China). Sequencing reads were compared to those in the Rfm database. The accession number of the reference genome was GCF_000203855.3. The transcriptome sequencing raw data in fastq format raw reads were deposited in the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) database (accession number: PRJNA751435).
8
Comprehensive 16S rRNA Sequence Analysis
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The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by Trimmomatic and merged by FLASH. Operational taxonomic units (OTUs) with 97% similarity cutoff were clustered using UPARSE (version 7.1, http://drive5.com/uparse/ ), and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier (http://rdp.cme.msu.edu/ ) against the SILVA 16S rRNA database (Release123) using the confidence threshold of 0.7. The analysis was performed using the free online platform, Majorbio I-sanger Cloud Platform (https:// www.i-sanger.com ).
9
Microbial 16S rRNA Gene Sequencing
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The sequence length of the V4 hypervariable region of 16S rRNA gene was approximately 250 base pairs for most microbial species. All paired‐end reads from the original DNA fragment were assigned to each sample according to the unique barcodes and then merged using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/ ) to obtain raw sequences (Magoc & Salzberg, 2011 ). Prior to getting the quality‐filtered reads, removed chimeric sequences in clean sequences using UCHIME algorithm software (http://www.drive5.com/usearch/manual/uchime_algo.html ) (Edgar, Haas, Clemente, Quince, & Knight, 2011 ) and Gold database (http://drive5.com/uchime/uchime_download.html ), low‐quality raw sequences were strictly filtered to generate clean sequences by QIIME software (Quantitative Insights into Microbial Ecology, version 1.7.0, http://qiime.org/scripts/split_libraries_fastq.html ) (Bokulich et al., 2013 ). Sequence data were deposited in the NCBI Sequence Read Archive (SRA) (Accession number: SRR9645031, SRR9645032, SRR9645033, SRR9645034, SRR9645035). The data were analyzed on the free online platform of Majorbio I‐Sanger Cloud Platform (http://www.i-sanger.com ).
10
Bioinformatic Analysis of Experimental Data
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The experimental data were expressed as ± SD. SPSS 26 software was used for statistical analysis, and Origin 2020b was used for drawing. After one-way analysis of variance (ANOVA), statistical analysis between groups was performed by Duncan’s multiple range tests, and the differences were considered as significance at p < 0.05. Bioinformatic analysis is carried out on Majorbio I-Sanger cloud platform.
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