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SARA module 3: NGS epitope sequencing: Illumina FASTQ files

Citation

Walker, Thomas; Megremis, Spyridon; Lamb, Janine (2021), SARA module 3: NGS epitope sequencing: Illumina FASTQ files, Dryad, Dataset, https://doi.org/10.5061/dryad.gmsbcc2mb

Abstract

We investigate the accumulated microbial and autoantigen antibody repertoire in adult-onset dermatomyositis patients sero-positive for TIF1γ (TRIM33) autoantibodies. We use an untargeted high-throughput approach which combines immunoglobulin disease-specific epitope-enrichment and identification of microbial and human antigens. We observe antibodies recognizing a wider repertoire of microbial antigens in dermatomyositis. Antibodies recognizing viruses and Poxviridae family species are significantly enriched. The identified autoantibodies recognise a large portion of the human proteome, including interferon regulated proteins; these proteins cluster in specific biological processes. In addition to TRIM33, we identify autoantibodies against eleven further TRIM proteins, including TRIM21. Some of these TRIM proteins share epitope homology with specific viral species including poxviruses. Our data suggest antibody accumulation in dermatomyositis against an expanded diversity of microbial and human proteins and evidence of non-random targeting of specific signalling pathways. Our findings indicate that molecular mimicry and epitope spreading events may play a role in dermatomyositis pathogenesis.

Methods

We implemented the “Serum Antibody Repertoire Analysis (SARA)” pipeline as outlined in our corresponding manuscript.

Polyvalent plasmid variance regions were PCR amplified and gel-purified. PCR products were validated by Sanger sequencing and NanoDrop. Multiplexed DNA fragments were sequenced on the NextSeq 500 platform (Illumina, UK) to retrieve ≈24 million and ≈36 million paired end FASTQ reads for HC and DM respectively.

This repository contains FASTQ zipped files.

FASTQ IDs are:

"S1_S1_": P20 pool Healthy controls (HC).

"S2_S2_": P20 pool: Anti-TIF1 positive Dermatomyositis patients (DM).

"S5_S5_": P10 pool: Healthy controls (HC).

"S6_S6_": P10 pool: Anti-TIF1 positive Dermatomyositis patients (DM).

Usage Notes

The FASTQ files contain plasmid sequence data following competitive biopanning of total Igs that were purified from healthy and disease plasma.

Plasmid sequences contain a 36bp variance region which codes for expressed (and thus immunologically-relevant) peptide epitopes. 

Funding

CHILDREN with CANCER UK

RCUK | Medical Research Council, Award: MR/N003322/1

The Myositis Association. The Caring Cancer Trust. The Humane Research Trust. Cancer Prevention Research Trust.