210 were aligned to the human genome (human_glk_v37) using HISAT (ref v0.1.6) (with maximum allowance of two mismatches), and read sorting was performed using SAMtools (ref v0.1.19). SAMtools flagstat and Picard tools (ref v2.9.0) were used to obtain mapping statistics. Six samples with low percentage read alignment (< 90%) were removed. Gene expression was estimated using HTSeq (ref v0.9.1), based on Ensemble version 75 annotation, resulting in a RNA expression dataset featuring 15,934 genes. Expression data on gene level were normalized using a trimmed mean of M values, and clr transformation was applied, resulting in 826 mucosal RNA-seq samples. Mucosal 16S rRNA gene sequencing Total DNA extraction of intestinal biopsies using 0.25 g of sample was performed as described previously, with minor modifications.100 Microbial composition of intestinal biopsies was determined by Illumina MiSeq paired-end sequencing of the V3-V4 hypervariable region of the 16S rRNA gene (MiSeq Benchtop Sequencer, Illumina Inc., San Diego, USA). Amplification of bacterial DNA was performed by PCR using modified 341F and 806R primers with a six-nucleotide barcode on the 806R primer for multiplexing.101,102 Sequences of both primers can be found in Supplementary Table S1. Both primers contain an Illumina MiSeq adapter sequence, which is necessary for flow cell–binding in the MiSeq machine. A detailed overview of the PCR, DNA cleanup and MiSeq library preparation using a 2x300 cartridge can be found in the Supplementary Methods. Read trimming and filtering was done using Trimmomatic (0.33) to obtain an average read quality of 25 and a minimum length of 50. Quality was further checked using R package DADA2 (v1.03) with the following parameters: truncLen=c(240,160), maxN=0, maxEE=c(2,2), truncQ=2 and rm.phix=TRUE. After error correction and chimera removal, the amplicon sequence variants were assigned to the silva database (v.132). Samples with >2,000mapped reads were used for further analysis, resulting in 755 mucosal 16S samples. After accounting for overlap between mucosal RNA-seq and mucosal 16S data, 696 intestinal biopsies from 337 different patients and 16 non-IBD controls were available for host–microbiota interaction analyses Chapter 6
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