108 consisted of virulence factors derived from the virulence factor database (VFDB) and ~5% (11,525 oligos) were (biological and technical) controls. Concerning the 100,000-variant library, 40,000 peptides represented phages, 30,000 allergens and 30,000 antigens derived from the Immune Epitope Database (IEDB).23 In general, antigens were rationally selected by prioritizing previously reported bacterial antigens and strains known to trigger antibody responses and potential uncharacterized antigens, while focusing on antigens having a high chance of being exposed to the host immune system, e.g. antigens from bacteria with high abundances, membrane proteins, secreted proteins and motility proteins. Selection of antigens was performed without thought of sequence length, necessitating splitting into peptides with a given limit for DNA oligonucleotide synthesis of 230 nt. Immunoprecipitation and next-generation sequencing Experimental procedures for immunoprecipitation (IP) and next-generation sequencing (NGS) were performed as described previously, but with minor modifications.12,15 Polymerase chain reaction (PCR) plates used for bead transfer and washing were blocked with 150 μL bovine serum albumin (BSA, 30 g/L in Dulbecco’s phosphate-buffered saline at 4°C overnight incubation). Next, BSA was added to the diluted phage/antibody mixtures for IP at a concentration of 2 g/L. Phage wash buffer that was used for IP contained 0.1 wt/vol% IPEGAL CA 630 (Sigma-Aldrich, catalog no. 13021). Subsequently, antibodies (3 μg of serum IgG antibodies as measured by ELISA) and phages (4,000-fold phage coverage per library variant) were mixed after optimization procedures that were performed to determine the optimal amounts of antibodies and phages for IP. The microbiota antigen library was mixed with a 200 nt 100,000 variant pool in a 2:1 ratio. 96-well plates were incubated with the phage/antibody mixtures at 4°C while mixed on an overhead rotator. Protein A and G magnetic beads were incubated overnight on a rotator at 4°C, and 40 μL of this mixture was added to the plates in a 1:1 ratio (Thermo-Fisher Scientific, catalog nos. 10008D and 10009D) according to manufacturer’s instructions. Beads were put into PCR plates and washed twice after 4-h incubation using a Tecan freedom Evo liquid-handling robot with filter tips. Pooled Illumina amplicon sequencing was performed with PCR amplifications using Q5 polymerase (New England Biolabs, catalog no. M0493L) (see Supplementary Methods for primer details), and PCR products were paired-end sequenced on an Illumina NextSeq machine. Metagenomics shotgun sequencing (MGS) of fecal samples Fecal metagenomics data were available from a subset of the IBD cohort taken within 1 year of serum sampling (n = 137) as well as from the LL-DEEP cohort.22 Patients and participants were requested to collect and freeze fecal samples at their homes, after which the samples were picked up, transported on dry ice and stored at -80°C until further analysis. Microbial taxonomy was determined with high-resolution whole-genome MGS. DNA extraction procedures and MGS sequencing with the Illumina HiSeq platform were performed as described previously.5,24 Genomic library preparation was conducted using the Nextera XT Library preparation kit. Removal of adapters and trimming of the ends of metagenomics reads was performed using Trimmomatic (v.0.32). Cleaned metagenomic reads were further processed through a bioinformatic pipeline as described previously.5,25 Taxonomic profiling was performed using MetaPhlAn3 with default Chapter 4
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