Biology subjects

Blackburn, J.

Publications and source records attributed to Blackburn, J..

4 recordsLinked to original sources

bioRxiv2018

High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes

High-throughput single-cell RNA-Sequencing is a powerful technique for gene expression profiling of complex and heterogeneous cellular populations such as the immune system. However, these methods only provide short-read sequence from one end of a cDNA template, making them poorly suited to the investigation of gene-regulatory events such as mRNA splicing, adaptive immune responses or somatic genome evolution. To address this challenge, we have developed a method that combines targeted long-read sequencing with short-read based transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning. We use Repertoire And Gene Expression sequencing (RAGE-seq) to accurately characterize full-length T cell (TCR) and B cell (BCR) receptor sequences and transcriptional profiles of more than 7,138 lymphocytes sampled from the primary tumour and draining lymph node of a breast cancer patient. With this method we show that somatic mutation, alternate splicing and clonal evolution of T and B lymphocytes can be tracked across these tissue compartments. Our results demonstrate that RAGE-Seq is an accessible and cost-effective method for high-throughput deep single cell profiling, applicable to a wide range of biological challenges.

genomics↗

bioRxiv2018

Chiral DNA sequences as commutable reference standards for clinical genomics

Chirality is a geometric property describing any object that is inequivalent to a mirror image of itself. Due to its 5-3 directionality, a DNA sequence is distinct from a mirrored sequence arranged in reverse nucleotide order, and is therefore chiral. A given sequence and its opposing chiral partner sequence share many properties, such as nucleotide composition and sequence entropy. Here we demonstrate that chiral DNA sequence pairs also perform equivalently during molecular and bioinformatic techniques that underpin modern genetic analysis, including PCR amplification, hybridization, whole-genome, target-enriched and nanopore sequencing, sequence alignment and variant detection. Given these shared properties, synthetic DNA sequences that directly mirror clinically relevant and/or analytically challenging regions of the human genome are ideal reference standards for clinical genomics. We show how the addition of chiral DNA standards to patient tumor samples can prevent false-positive and false-negative mutation detection and, thereby, improve diagnosis. Accordingly, we propose that chiral DNA standards can fulfill the unmet need for commutable internal reference standards in precision medicine.

genomics↗

bioRxiv2017

Spores and soil from six sides: interdisciplinarity and the environmental biology of anthrax (Bacillus anthracis)

Environmentally Transmitted Diseases Are Comparatively Poorly Understood And Managed, And Their Ecology Is Particularly Understudied. Here We Identify Challenges Of Studying Environmental Transmission And Persistence With A Six-Sided Interdisciplinary Review Of The Biology Of Anthrax (Bacillus Anthracis). Anthrax Is A Zoonotic Disease Capable Of Maintaining Infectious Spore Banks In Soil For Decades (Or Even Potentially Centuries), And The Mechanisms Of Its Environmental Persistence Have Been The Topic Of Significant Research And Controversy. Where Anthrax Is Endemic, It Plays An Important Ecological Role, Shaping The Dynamics Of Entire Herbivore Communities. The Complex Eco-Epidemiology Of Anthrax, And The Mysterious Biology Of Bacillus Anthracis During Its Environmental Stage, Have Necessitated An Interdisciplinary Approach To Pathogen Research. Here, We Illustrate Different Disciplinary Perspectives Through Key Advances Made By Researchers Working In Etosha National Park, A Long-Term Ecological Research Site In Namibia That Has Exemplified The Complexities Of AnthraxS Enzootic Process Over Decades Of Surveillance. In Etosha, The Role Of Scavengers And Alternate Routes (Waterborne Transmission And Flies) Has Proved Unimportant, Relative To The Long-Term Persistence Of Anthrax Spores In Soil And Their Infection Of Herbivore Hosts. Carcass Deposition Facilitates Green-Ups Of Vegetation To Attract Herbivores, Potentially Facilitated By Anthrax Spores Role In The Rhizosphere. The Underlying Seasonal Pattern Of Vegetation, And Herbivores Immune And Behavioral Responses To Anthrax Risk, Interact To Produce Regular \"Anthrax Seasons\" That Appear To Be A Stable Feature Of The Etosha Ecosystem. Through The Lens Of Microbiologists, Geneticists, Immunologists, Ecologists, Epidemiologists, And Clinicians, We Discuss How Anthrax Dynamics Are Shaped At The Smallest Scale By Population Genetics And Interactions Within The Bacterial Communities Up To The Broadest Scales Of Ecosystem Structure. We Illustrate The Benefits And Challenges Of This Interdisciplinary Approach To Disease Ecology, And Suggest Ways Anthrax Might Offer Insights Into The Biology Of Other Important Pathogens. Bacillus Anthracis, And The More Recently Emerged Bacillus Cereus Biovar Anthracis, Share Key Features With Other Environmentally-Transmitted Pathogens, Including Several Zoonoses And Panzootics Of Special Interest For Global Health And Conservation Efforts. Understanding The Dynamics Of Anthrax, And Developing Interdisciplinary Research Programs That Explore Environmental Persistence, Is A Critical Step Forward For Understanding These Emerging Threats.

ecology↗

bioRxiv2017

Universal Alternative Splicing Of Noncoding Exons

The human transcriptome is so large, diverse and dynamic that, even after a decade of investigation by RNA sequencing (RNA-Seq), we are yet to resolve its true dimensions. RNA-Seq suffers from an expression-dependent bias that impedes characterization of low-abundance transcripts. We performed targeted single-molecule and short-read RNA-Seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome, identifying a fundamental distinction between protein-coding and noncoding gene content: almost every noncoding exon undergoes alternative splicing, producing a seemingly limitless variety of isoforms. Analysis of syntenic regions of the mouse genome shows that few noncoding exons are shared between human and mouse, yet human splicing profiles are recapitulated on Hsa21 in mouse cells, indicative of regulation by a deeply conserved splicing code. We propose that noncoding exons are functionally modular, with alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution.

genomics↗