Biology subjects

Gamble, V.

Publications and source records attributed to Gamble, V..

3 recordsLinked to original sources

bioRxiv2024

Anchor-Enhanced Bead Design for Reduced Oligonucleotide Synthesis Errors in Single-cell sequencing

Single-cell transcriptomics, reliant on the incorporation of barcodes and unique molecular identifiers (UMIs) into captured polyA+ mRNA, faces a significant challenge due to synthesis errors in oligonucleotide capture sequences. These inaccuracies, which are especially problematic in long-read sequencing, impair the precise identification of sequences and result in inaccuracies in UMI deduplication. To mitigate this issue, we have modified the oligonucleotide capture design, which integrates an interposed anchor between the barcode and UMI, and a V base anchor adjacent to the polyA capture region. This configuration is devised to ensure compatibility with both short and long-read sequencing technologies, facilitating improved UMI recovery and enhanced feature detection, thereby improving the efficacy of droplet-based sequencing methods.

molecular biology↗

bioRxiv2024

A systems-based approach to uterine fibroids identifies differential splicing associated with abnormal uterine bleeding

Uterine fibroids (UFs), benign tumours prevalent in up to 80% of women of reproductive age, are associated with significant morbidity, including abnormal uterine bleeding, pain and infertility. Despite identification of key genomic alterations in MED12 and HMGA2, the pathogenic mechanisms underlying UFs and heavy menstrual bleeding (HMB) remain poorly understood. To correlate systematically genetic, transcriptional and proteomic phenotypes, our study involved an integrative analysis of fibroid, myometrium and endometrium tissues from 137 patients, utilising genome-wide SNP arrays, targeted sequencing, RNA sequencing and proteomics. Our findings reveal 39.7% of UFs possess MED12 mutations, alongside novel variants in genes such as COL4A5 and COL4A6. Multi-omics factor analysis of integrated protein and mRNA highlighted differential regulation related to extracellular matrix remodelling, proteolysis and homeostasis in fibroid versus myometrium tissues, and distinct gene sets associated with RNA splicing in the endometrium of patients with HMB, particularly in MED12-mutated fibroids. Our study proposes a model, which is supported by in vivo evidence, where altered signalling of MED12-mutated fibroids influences RNA transcript isoform expression in endometrium, potentially leading to abnormal uterine bleeding. This integrative approach unravels complex molecular pathways in UF pathogenesis and HMB, offering novel insights for targeted therapeutic development.

systems biology↗

bioRxiv2023

Correcting PCR amplification errors in unique molecular identifiers to generate absolute numbers of sequencing molecules.

Unique Molecular Identifiers (UMIs) are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are the main source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing UMIs using homotrimeric nucleotide blocks provides an error correcting solution, that allows absolute counting of sequenced molecules.

genomics↗