The sequence of sequencers: The history of sequencing DNA

Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way

T-cell receptor repertoire sequencing in health and disease

The adaptive immune systems of jawed vertebrates are based upon lymphocytes bearing a huge variety of antigen receptors. Produced by somatic DNA recombination, these receptors are clonally expressed on T- and B-lymphocytes, where they are used to help detect and control infections and help maintain regular bodily function. Full understanding of various aspects of the immune system relies upon accurate measurement of the individual receptors that make up these repertoires. In order to obtain such data, protocols were developed to permit unbiased amplification, high-throughput deep-sequencing, and error-correcting bioinformatic analysis of T-cell receptor sequences. These techniques have been applied to peripheral blood samples to further characterise aspects of the TCR repertoire of healthy individuals, such as V(D)J TCR gene usage and pairing distributions. A large number of sequences are also found to be shared across multiple individuals, including sequences matching receptors belonging to known and proposed T-cell subsets making use of invariant rearrangements. The resolution provided also permitted detection of low-frequency recombination events that use unexpected gene segments, or contained alternative splicing events. Deep-sequencing was further used to study the effect of HIV infection, and subsequent antiretroviral therapy, upon the TCR repertoire. HIV-patient repertoires are typified by marked clonal inequality and perturbed population structures, relative to healthy controls. The data presented support a model in which HIV infection drives expansion of an subset of CD8+ clones, which -- in combination with the virally-mediated loss of CD4+ cells -- is responsible for driving repertoires towards an idiosyncratic population with low diversity. Moreover these altered repertoire features do not significantly recover after three months of therapy. Deep-sequencing therefore presents opportunities to investigate the properties of TCR repertoires both in health and disease, which could be useful when analysing a wide variety of immune phenomena

Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding

The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq, and in the context of analysis of T and B cell receptor repertoires. In this study, we combine DNA barcoding with HTS to quantify PCR output from individual target molecules. We develop computational tools that simulate both the PCR branching process itself, and the subsequent subsampling which typically occurs during HTS sequencing. We explore the influence of different types of heterogeneity on sequencing output, and compare them to experimental results where the efficiency of amplification is measured by barcodes uniquely identifying each molecule of starting template. Our results demonstrate that the PCR process introduces substantial amplification heterogeneity, independent of primer sequence and bulk experimental conditions. This heterogeneity can be attributed both to inherited differences between different template DNA molecules, and the inherent stochasticity of the PCR process. The results demonstrate that PCR heterogeneity arises even when reaction and substrate conditions are kept as constant as possible, and therefore single molecule barcoding is essential in order to derive reproducible quantitative results from any protocol combining PCR with HTS

Dynamic Perturbations of the T-Cell Receptor Repertoire in Chronic HIV Infection and following Antiretroviral Therapy

HIV infection profoundly affects many parameters of the immune system and ultimately leads to AIDS, yet which factors are most important for determining resistance, pathology, and response to antiretroviral treatment - and how best to monitor them - remain unclear. We develop a quantitative high-throughput sequencing pipeline to characterize the TCR repertoires of HIV-infected individuals before and after antiretroviral therapy, working from small, unfractionated samples of peripheral blood. This reveals the TCR repertoires of HIV(+) individuals to be highly perturbed, with considerably reduced diversity as a small proportion of sequences are highly overrepresented. HIV also causes specific qualitative changes to the repertoire including an altered distribution of V gene usage, depletion of public TCR sequences, and disruption of TCR networks. Short-term antiretroviral therapy has little impact on most of the global damage to repertoire structure, but is accompanied by rapid changes in the abundance of many individual TCR sequences, decreases in abundance of the most common sequences, and decreases in the majority of HIV-associated CDR3 sequences. Thus, high-throughput repertoire sequencing of small blood samples that are easy to take, store, and process can shed light on various aspects of the T-cell immune compartment and stands to offer insights into patient stratification and immune reconstitution

Phages in the Gut Ecosystem

Phages, short for bacteriophages, are viruses that specifically infect bacteria and are the most abundant biological entities on earth found in every explored environment, from the deep sea to the Sahara Desert. Phages are abundant within the human biome and are gaining increasing recognition as potential modulators of the gut ecosystem. For example, they have been connected to gastrointestinal diseases and the treatment efficacy of Fecal Microbiota Transplant. The ability of phages to modulate the human gut microbiome has been attributed to the predation of bacteria or the promotion of bacterial survival by the transfer of genes that enhance bacterial fitness upon infection. In addition, phages have been shown to interact with the human immune system with variable outcomes. Despite the increasing evidence supporting the importance of phages in the gut ecosystem, the extent of their influence on the shape of the gut ecosystem is yet to be fully understood. Here, we discuss evidence for phage modulation of the gut microbiome, postulating that phages are pivotal contributors to the gut ecosystem dynamics. We therefore propose novel research questions to further elucidate the role(s) that they have within the human ecosystem and its impact on our health and well-being

Feature selection using a one dimensional naïve Bayes' classifier increases the accuracy of support vector machine classification of CDR3 repertoires.

MOTIVATION: Somatic DNA recombination, the hallmark of vertebrate adaptive immunity, has the potential to generate a vast diversity of antigen receptor sequences. How this diversity captures antigen specificity remains incompletely understood. In this study we use high throughput sequencing to compare the global changes in T cell receptor β chain complementarity determining region 3 (CDR3β) sequences following immunization with ovalbumin administered with complete Freund's adjuvant (CFA) or CFA alone. RESULTS: The CDR3β sequences were deconstructed into short stretches of overlapping contiguous amino acids. The motifs were ranked according to a one-dimensional Bayesian classifier score comparing their frequency in the repertoires of the two immunization classes. The top ranking motifs were selected and used to create feature vectors which were used to train a support vector machine. The support vector machine achieved high classification scores in a leave-one-out validation test reaching  : >90% in some cases. SUMMARY: The study describes a novel two-stage classification strategy combining a one-dimensional Bayesian classifier with a support vector machine. Using this approach we demonstrate that the frequency of a small number of linear motifs three amino acids in length can accurately identify a CD4 T cell response to ovalbumin against a background response to the complex mixture of antigens which characterize Complete Freund's Adjuvant. AVAILABILITY AND IMPLEMENTATION: The sequence data is available at www.ncbi.nlm.nih.gov/sra/?term¼SRP075893 The Decombinator package is available at github.com/innate2adaptive/Decombinator The R package e1071 is available at the CRAN repository https://cran.r-project.org/web/packages/e1071/index.html CONTACT: [email protected] information: Supplementary data are available at Bioinformatics online

Factors associated with alcohol reduction in harmful and hazardous drinkers following alcohol brief intervention in Scotland: a qualitative enquiry

Background: Alcohol Brief Intervention (ABI) uses a motivational counselling approach to support individuals to reduce excessive alcohol consumption. There is growing evidence on ABI’s use within various health care settings, although how they work and which components enhance success is largely unknown. This paper reports on the qualitative part of a mixed methods study. It explores enablers and barriers associated with alcohol reduction following an ABI. It focuses on alcohol’s place within participants’ lives and their personal perspectives on reducing consumption. There are a number of randomised controlled trials in this field though few ABI studies have addressed the experiences of hazardous/harmful drinkers. This study examines factors associated with alcohol reduction in harmful/hazardous drinkers following ABI. Methods: This qualitative study was underpinned by a realist evaluation approach and involved semistructured interviews with ten harmful or hazardous alcohol drinkers. Participants (n = 10) were from the intervention arm of a randomised controlled trial (n = 124). All had received ABI, a 20 min motivational counselling interview, six months previously, and had reduced their alcohol consumption. Interviews were recorded, transcribed verbatim and thematically analysed. Results: Participants described their views on alcohol, its’ place in their lives, their personal perspectives on reducing their consumption and future aspirations. Conclusions: The findings provide an insight into participants’ views on alcohol, ABI, and the barriers and enablers to change. Participants described a cost benefit analysis, with some conscious consideration of the advantages and disadvantages of reducing intake or abstaining from alcohol. Findings suggest that, whilst hospital admission can act as a catalyst, encouraging individuals to reflect on their alcohol consumption through ABI may consolidate this, turning this reflective moment into action. Sustainability may be enhanced by the presence of a ‘significant other’ who encourages and experiences benefit. In addition having a purpose or structure with activities linked to employment and/or social and leisure pursuits offers the potential to enhance and sustain reduced alcohol consumption. Trial registration: Trial registration number TRN NCT00982306 September 22nd 200

Specificity, Privacy, and Degeneracy in the CD4 T Cell Receptor Repertoire Following Immunization.

T cells recognize antigen using a large and diverse set of antigen-specific receptors created by a complex process of imprecise somatic cell gene rearrangements. In response to antigen-/receptor-binding-specific T cells then divide to form memory and effector populations. We apply high-throughput sequencing to investigate the global changes in T cell receptor sequences following immunization with ovalbumin (OVA) and adjuvant, to understand how adaptive immunity achieves specificity. Each immunized mouse contained a predominantly private but related set of expanded CDR3β sequences. We used machine learning to identify common patterns which distinguished repertoires from mice immunized with adjuvant with and without OVA. The CDR3β sequences were deconstructed into sets of overlapping contiguous amino acid triplets. The frequencies of these motifs were used to train the linear programming boosting (LPBoost) algorithm LPBoost to classify between TCR repertoires. LPBoost could distinguish between the two classes of repertoire with accuracies above 80%, using a small subset of triplet sequences present at defined positions along the CDR3. The results suggest a model in which such motifs confer degenerate antigen specificity in the context of a highly diverse and largely private set of T cell receptors

3D multi-agent models for protein release from PLGA spherical particles with complex inner morphologies

In order to better understand and predict the release of proteins from bioerodible micro- or nanospheres, it is important to know the influences of different initial factors on the release mechanisms. Often though it is difficult to assess what exactly is at the origin of a certain dissolution profile. We propose here a new class of fine-grained multi-agent models built to incorporate increasing complexity, permitting the exploration of the role of different parameters, especially that of the internal morphology of the spheres, in the exhibited release profile. This approach, based on Monte-Carlo (MC) and Cellular Automata (CA) techniques, has permitted the testing of various assumptions and hypotheses about several experimental systems of nanospheres encapsulating proteins. Results have confirmed that this modelling approach has increased the resolution over the complexity involved, opening promising perspectives for future developments, especially complementing in vitro experimentation