4 research outputs found

    Comparison of T-cell Receptor Diversity of people with Myalgic Encephalomyelitis versus controls

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    Objective: Myalgic Encephalomyelitis (ME; sometimes referred to as Chronic Fatigue Syndrome) is a chronic disease without laboratory test, detailed aetiological understanding or effective therapy. Its symptoms are diverse, but it is distinguished from other fatiguing illnesses by the experience of post-exertional malaise, the worsening of symptoms even after minor physical or mental exertion. Its frequent onset after infection suggests autoimmune involvement or that it arises from abnormal T-cell activation. Results: To test this hypothesis, we sequenced the genomic loci of and T-cell receptors (TCR) from 40 human blood samples from each of four groups: severely affected people with ME; mildly or moderately affected people with ME; people diagnosed with Multiple Sclerosis, as disease controls; and, healthy controls. Seeking to automatically classify these individuals’ samples by their TCR repertoires, we applied P-SVM, a machine learning method. However, despite working well on a simulated data set, this approach did not allow statistically significant partitioning of samples into the four subgroups. Our findings do not support the hypothesis that blood samples from people with ME frequently contain altered T-cell receptor diversity

    Violation of the 12/23 rule of genomic V(D)J recombination is common in lymphocytes

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    V(D)J genomic recombination joins single gene segments to encode an extensive repertoire of antigen receptor specificities in T and B lymphocytes. This process initiates with double-stranded breaks adjacent to conserved recombination signal sequences that contain either 12- or 23-nucleotide spacer regions. Only recombination between signal sequences with unequal spacers results in productive coding genes, a phenomenon known as the '12/23 rule.' Here we present two novel genomic tools that allow the capture and analysis of immune locus rearrangements from whole thymic and splenic tissues using second-generation sequencing. Further, we provide strong evidence that the 12/23 rule of genomic recombination is frequently violated under physiological conditions, resulting in unanticipated hybrid recombinations in ∼10% of Tcra excision circles. Hence, we demonstrate that strict adherence to the 12/23 rule is intrinsic neither to recombination signal sequences nor to the catalytic process of recombination and propose that nonclassical excision circles are liberated during the formation of antigen receptor diversity

    Preparation of high-quality next-generation sequencing libraries from picogram quantities of target DNA

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    New sequencing technologies can address diverse biomedical questions but are limited by a minimum required DNA input of typically 1 μg. We describe how sequencing libraries can be reproducibly created from 20 pg of input DNA using a modified transpososome-mediated fragmentation technique. Resulting libraries incorporate in-line bar-coding, which facilitates sample multiplexes that can be sequenced using Illumina platforms with the manufacturer's sequencing primer. We demonstrate this technique by providing deep coverage sequence of the Escherichia coli K-12 genome that shows equivalent target coverage to a 1-μg input library prepared using standard Illumina methods. Reducing template quantity does, however, increase the proportion of duplicate reads and enriches coverage in low-GC regions. This finding was confirmed with exhaustive resequencing of a mouse library constructed from 20 pg of gDNA input (about seven haploid genomes) resulting in ∼0.4-fold statistical coverage of uniquely mapped fragments. This implies that a near-complete coverage of the mouse genome is obtainable with this approach using 20 genomes as input. Application of this new method now allows genomic studies from low mass samples and routine preparation of sequencing libraries from enrichment procedures
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