Additional file 2 of Profiling non-coding RNA levels with clinical classifiers in pediatric Crohn’s disease
Creators: name: Pelia, Ranjit
name: Venkateswaran, Suresh
name: Matthews, Jason D.
name: Haberman, Yael
name: Cutler, David J.
name: Hyams, Jeffrey S.
name: Denson, Lee A.
name: Kugathasan, Subra
Publisher: name: figshare
Year: 2021
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Physiology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Ecology
subject: Immunology
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 2: Fig. S1. Overall Workflow of Analysis: Differential expression analysis was performed on n = 345 ileal and n = 390 rectal Bulk-RNA-later biopsies using Illumina deep-RNA sequencing in a single batch. The reference panel utilized was Hg38 with STAR package used for alignment of 58,381 transcripts, of which n = 20,779 were ncRNAs. EdgeR was used for differential expression of these ncRNAs in three comparisons: Disease Status, Disease Behavior, and Disease Inflammation. For each analysis, the total number of DE ncRNAs for ileal and rectal datasets is represented. Fig. S2. a The log2FC obtained from case-control analysis of all the 20,779 ncRNAs were compared between ileal and rectal biopsies. Each point is a ncRNA expression and they colored based on tissue- or disease-specific. b Using a subset of newly diagnosed CD patients (n = 133) from the ileal dataset (n = 345), the DE analysis results were compared with our previous study (Haberman et al. 2018) results performed on the same RISK cohort. The strong positive log2FC results shows the reliability and replicability of our analysis. Fig. S3. Gene Ontology Analysis of Crohn’s Disease versus Controls DE ncRNAs in Ileal Biopsies: Using TopGO, gene ontology analysis was conducted on n = 89 DE ncRNAs in ileal biopsies. The results displayed significant, FDR &lt; 0.05, hits in (a) cellular components (n = 79), and b biological processes (n = 21). Fig. S4. Gene Ontology Analysis of Crohn’s Disease versus Controls DE ncRNAs in Rectal Biopsies: Using TopGO, gene ontology analysis was conducted on n = 89 DE ncRNAs in rectal biopsies. The results displayed significant, FDR &lt; 0.05, hits in a cellular components (n = 12), and b biological processes (n = 17). Fig. S5. Principal Components and Volcano Plots of DE ncRNAs based on Crohn’s Disease Behavior: The PCs were calculated using entire list of ncRNA (n = 20,779) and first two PCs were plotted. Each point represents a subject and they are clearly separating the CD disease severity groups from controls across a B1 versus controls, b B2 versus Controls, and c B3 versus Controls, in ileal biopsies. The volcano plots of DE analysis show DE ncRNAs were identified across d B1 versus controls (n = 70), e B2 versus controls (n = 124), f and B3 versus controls (n = 22) comparisons. Fig. S6. Types of ncRNAs observed in Crohn’s Disease Behavior DE ncRNAs: Sankey plots of DE ncRNAs observed in ileal samples by comparing a B2 versus B1 (n = 35), and b B3 versus B2 (n=14) were correlated with protein coding genes (n=18,927) from the same dataset. Those pairs of ncRNA-mRNA with correlations &gt; 0.50 were deemed significant. Using these correlation pairs, the relationship of ncRNAs and mRNAs were sequestered based on CIS, same strand, or TRANS, different strands. The coding strand is represented by (−) and non-coding strand by (+). Fig. S7. Principal Component Analysis of CD patients available with Inflammation Status ncRNAs: The disease inflammation location classifiers: L1, L2, L3, were used to classify the inflammation status. Any CD patients had disease at L1, L2 and L3 were considered as inflamed and the rest were considered as non-inflamed samples. The first two PCs were plotted to see any clusters based on inflammation status in (a) ileal biopsies (n=345) and (b) rectal (n = 390) rectal biopsies. Fig. S8. Principal Component Analysis of Inflammation Location ncRNAs: Using all n = 20,779 ncRNAs, princomp was used to extrapolate PCs in order to visualize trends amongst disease inflammation location groups in entire CD patients. PCs were calculated for inflamed versus non-inflamed samples a Both L1 + L3 (n = 198) CD patients with ileal disease were considered as inflamed group and compared against the non-inflamed group (n = 20 b), and non-inflammed + L2 (n = 76) group, because the L2 CD patients are inflamed in colonic region (c).