E-MTAB-9746
RNA-seq of biopsies from the colons of patients with Crohn's Disease classified as healed versus non-healed according to different classification scores
The objective of the study was to understand gene expression patterns and pathways that relate to Endoscopic Remission and Histologic remission in IBD as defined by different endoscopic and histological disease activity scores.    Colonic Endoscopic biopsies were collected in RNAlater from 9 Crohn's disease  patients undergoing colonoscopies with the endocytoscope CF- Y-0058-1 prototype Olympus, Japan). RNA was extracted using RNeasy columns (Qiagen) and quantified by Qubit (Life Technologies).  0.5ng RNA was used to prepare uniquely indexed cDNA QIAseq UPX 3' Transcriptome libraries according to manufacturer's instructions (Qiagen). Libraries were quantified and quality-controlled using the QIAseq Library Quant Assay Kit and tapestation analysis and sequenced on the Miseq and Nextseq Illumina platforms using the illumina sequencing primer and PhiX 15% to a depth of 1-3milion reads/sample. Read numbers were set as 101 bp (R1) x 51 bp (R2). Fastq files were obtained through BaseSpace and reads de-multiplexed, aligned to human genome GRCh38, quantified and normalised by the TPM method using the CLC Genomics Workbench (Qiagen).   The following differential expression procedure was performed for participants classified as healed versus non-healed according different histologic and endoscpic scores which included the histological scores:  Nancy and Robarts Histological Index (RHI) as well as SES-CD, Modified Riley and and the endocytoscopy score system (ECSS).   Normalised values were log-transformed with a pseudocount of 1 added. Transcripts with < 1 transformed count in any sample were excluded from further analysis, as were transcripts with low variance (defined as less than 10% unique counts across both conditions and greater than a 19:1 ratio of the most frequent count to the second most frequent across both conditions). Differential expression analysis between conditions was conducted with the limma package. Library size was estimated using reduced maximum likelihood estimator with 500 iterations. Initial fitting was performed using a robust M-estimation, and moderated test statistics computed by empirical Bayes. A FDR corrected p value <0.05 was considered and filtered for further downstream data analysis. Partial least squares discriminant analysis (PLS-DA) modelling was performed on these genes and a variable Importance in Projection (VIP) score of >1 was used to further filter the genes. To understand the biological significance and pathways, we also performed enrichment analysis using EnrichR involving three databases: Gene ontology, Wiki pathways and Kyoto Encyclopaedia of Genes and Genomes (KEGG).
The objective of the study was to understand gene expression patterns and pathways that relate to Endoscopic Remission and Histologic remission in IBD as defined by different endoscopic and histological disease activity scores.    Colonic Endoscopic biopsies were collected in RNAlater from 9 Crohn's disease  patients undergoing colonoscopies with the endocytoscope CF- Y-0058-1 prototype Olympus, Japan). RNA was extracted using RNeasy columns (Qiagen) and quantified by Qubit (Life Technologies).  0.5ng RNA was used to prepare uniquely indexed cDNA QIAseq UPX 3' Transcriptome libraries according to manufacturer's instructions (Qiagen). Libraries were quantified and quality-controlled using the QIAseq Library Quant Assay Kit and tapestation analysis and sequenced on the Miseq and Nextseq Illumina platforms using the illumina sequencing primer and PhiX 15% to a depth of 1-3milion reads/sample. Read numbers were set as 101 bp (R1) x 51 bp (R2). Fastq files were obtained through BaseSpace and reads de-multiplexed, aligned to human genome GRCh38, quantified and normalised by the TPM method using the CLC Genomics Workbench (Qiagen).   The following differential expression procedure was performed for participants classified as healed versus non-healed according different histologic and endoscpic scores which included the histological scores:  Nancy and Robarts Histological Index (RHI) as well as SES-CD, Modified Riley and and the endocytoscopy score system (ECSS).   Normalised values were log-transformed with a pseudocount of 1 added. Transcripts with < 1 transformed count in any sample were excluded from further analysis, as were transcripts with low variance (defined as less than 10% unique counts across both conditions and greater than a 19:1 ratio of the most frequent count to the second most frequent across both conditions). Differential expression analysis between conditions was conducted with the limma package. Library size was estimated using reduced maximum likelihood estimator with 500 iterations. Initial fitting was performed using a robust M-estimation, and moderated test statistics computed by empirical Bayes. A FDR corrected p value <0.05 was considered and filtered for further downstream data analysis. Partial least squares discriminant analysis (PLS-DA) modelling was performed on these genes and a variable Importance in Projection (VIP) score of >1 was used to further filter the genes. To understand the biological significance and pathways, we also performed enrichment analysis using EnrichR involving three databases: Gene ontology, Wiki pathways and Kyoto Encyclopaedia of Genes and Genomes (KEGG).