107 PhIP-Seq microbiota antigen library Detailed information on the design, cloning and content of the PhIP-Seq microbiota antigen library (covering 244,000 peptide sequences) can be found in a recently published study.12 This microbiota antigen library was measured in combination with a second antigen library covering an additional 100,000 peptide sequences related to phages, viruses and immune epitopes.23 Figure 1 shows a schematic overview of the PhIP-Seq procedure and antigen library content. Antigen processing and microbiota phage library cloning Antigens incorporated into the microbiota phage library were cut into peptides with a maximum length of 64 amino acids (aa) (for the 100,000-variant library: 54 aa) with 20 aa overlap for adjoining peptides. To abide with original codon usage frequencies, antigen sequences were backtranslated to DNA using codon usage of E. coli (of highly expressed proteins), where restriction sites for cloning within the coding sequence were excluded. If required, coding was repeated in order to achieve unique barcodes in the coding sequence at the 44/75-nucleotide (nt) at the 3’-terminus of each oligonucleotide. As each barcode is a unique sequence (at Hamming distance three (44-nt) or five (75-nt) from all prior sequences in the library), a single read error while sequencing 44-nt barcodes can be corrected, while two read errors can be corrected while sequencing 75-nt barcodes. In addition, a part of the 5’-terminus was sequenced to exclude presence of multiple inserts by verifying the matching of 3’- and 5’-sequences. Alternate codons were randomly applied next to E. coli codon usage to allow discrimination between rather similar antigen sequences. Sequencing barcodes were incorporated into the coding sequence to enable usage of the entire oligonucleotide for encoding an antigen, rendering sequencing of the full coding sequence unnecessary. Antigen sequences shorter than 64 aa were encoded by adding a random sequence after the stop codon. Library amplification was initiated by adding the EcoRI and HindIII restriction sites, antigen sequences, stop codons and annealing sequences together as a 230nt-oligomer pool for the 244,000-variant library (Agilent Technologies, Santa Clara, CA, USA; see Supplementary Methods for primer details) and 200nt oligos for the 100,000-variant library (Twist Bioscience, San Francisco, CA, USA). Cloning of the antigen library into the T7 phages was performed following manufacturer’s instructions (T7Select 10-3 cloning kit, catalog no. 70550-3, Merck-Millipore, Burlington, MA, USA). Composition and design of the phage antigen libraries Two recently developed phage antigen libraries were used in this study, covering a total of 344,000 antigen peptides.12,23 One library consists primarily of microbiota antigens (covering 244,000 peptide sequences from 28,668 different proteins, of which 27,837 proteins were derived from microbiota antigens (excluding human proteins and controls)), while the other library includes phage, viral and food antigens (covering 100,000 antigen variants).23 Details on the origin and composition of the first library are described in the Supplementary Methods. Briefly, about 60% (147,061 oligos) of the microbiota antigen library was dedicated to bacterial genes and strains (122,551 oligos, ~50%, and 24,510 oligos, ~10%, respectively); 25% of the library (61,250 oligos) was represented by pathogenic bacterial species (24,500 oligos, ~10%), antibody-coated bacterial species (22,050 oligos, ~9%) and probiotic bacteria (14,700 oligos, ~6%); 10% (24,164 oligos) The antibody epitope repertoire in IBD
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