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

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

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

The effect of distance on reaction time in aiming movements

Target distance affects movement duration in aiming tasks but its effect on reaction time (RT) is poorly documented. RT is a function of both preparation and initiation. Experiment 1 pre-cued movement (allowing advanced preparation) and found no influence of distance on RT. Thus, target distance does not affect initiation time. Experiment 2 removed pre-cue information and found that preparing a movement of increased distance lengthens RT. Experiment 3 explored movements to targets of cued size at non-cued distances and found size altered peak speed and movement duration but RT was influenced by distance alone. Thus, amplitude influences preparation time (for reasons other than altered duration) but not initiation time. We hypothesise that the RT distance effect might be due to the increased number of possible trajectories associated with further targets: a hypothesis that can be tested in future experiments

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

Presymptomatic risk assessment for chronic non-communicable diseases

The prevalence of common chronic non-communicable diseases (CNCDs) far overshadows the prevalence of both monogenic and infectious diseases combined. All CNCDs, also called complex genetic diseases, have a heritable genetic component that can be used for pre-symptomatic risk assessment. Common single nucleotide polymorphisms (SNPs) that tag risk haplotypes across the genome currently account for a non-trivial portion of the germ-line genetic risk and we will likely continue to identify the remaining missing heritability in the form of rare variants, copy number variants and epigenetic modifications. Here, we describe a novel measure for calculating the lifetime risk of a disease, called the genetic composite index (GCI), and demonstrate its predictive value as a clinical classifier. The GCI only considers summary statistics of the effects of genetic variation and hence does not require the results of large-scale studies simultaneously assessing multiple risk factors. Combining GCI scores with environmental risk information provides an additional tool for clinical decision-making. The GCI can be populated with heritable risk information of any type, and thus represents a framework for CNCD pre-symptomatic risk assessment that can be populated as additional risk information is identified through next-generation technologies.Comment: Plos ONE paper. Previous version was withdrawn to be updated by the journal's pdf versio

Quantitative Characterization of the T Cell Receptor Repertoire of Naive and Memory subsets Using an Integrated experimental and Computational Pipeline Which Is Robust, economical, and Versatile

The T cell receptor (TCR) repertoire can provide a personalized biomarker for infectious and non-infectious diseases. We describe a protocol for amplifying, sequencing, and analyzing TCRs which is robust, sensitive, and versatile. The key experimental step is ligation of a single-stranded oligonucleotide to the 3′ end of the TCR cDNA. This allows amplification of all possible rearrangements using a single set of primers per locus. It also introduces a unique molecular identifier to label each starting cDNA molecule. This molecular identifier is used to correct for sequence errors and for effects of differential PCR amplification efficiency, thus producing more accurate measures of the true TCR frequency within the sample. This integrated experimental and computational pipeline is applied to the analysis of human memory and naive subpopulations, and results in consistent measures of diversity and inequality. After error correction, the distribution of TCR sequence abundance in all subpopulations followed a power law over a wide range of values. The power law exponent differed between naïve and memory populations, but was consistent between individuals. The integrated experimental and analysis pipeline we describe is appropriate to studies of T cell responses in a broad range of physiological and pathological contexts