Microbial and autoantibody immunogenic repertoires in TIF1? autoantibody positive dermatomyositis

Spyridon Megremis; Thomas Walker; Xiaotong He; James O'Sullivan; William E.R. Ollier; Hector Chinoy; Neil Pendleton; Antony Payton; Lynne Hampson; Ian Hampson; Janine Lamb

Forward: 5'-ATTCACCTGACTGACGAC-3', FliTrxTM Reverse: 5'-CCCTGATATTCGTCAGCG-3'. Multiplexed DNA fragments were sequenced on the NextSeq 500 platform (Illumina, UK). We retrieved ≈24 million and ≈36 million paired end FASTQ reads for HC and DM respectively (Figure 1 ). The 36-bp variance region sequences were translated with respect to reading frame. < 14% of DM and <13% of HC comprised premature stop codons, indicative of immunologically-relevant bio-panning enrichment ( Figure S1A -S1D). Non-specific HTS sequence noise from residual bio-panning solution was controlled for by our 10-σ-99 noise floor. This retains all expressed epitopes within each respective HC or DM pool that surpass a threshold of 10 standard deviations above the lowest 99% distinct peptide sequence enrichment when ranked by read count (further described in Supplementary figure S1A-S1D).