The design, manufacture and LNP formulation of mRNA for research use.

The delivery of mRNA provides a versatile platform to achieve rapid and robust protein expression within cells. mRNA delivery has therefore been widely adopted in research, and mRNA medicines are now being developed to treat a range of diseases. However, high-quality mRNA must be produced to ensure the safety, performance and effectiveness of these medicines.

A universal molecular control for DNA, mRNA and protein expression.

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression.

mRNA vaccine quality analysis using RNA sequencing.

The success of mRNA vaccines has been realised, in part, by advances in manufacturing that enabled billions of doses to be produced at sufficient quality and safety. However, mRNA vaccines must be rigorously analysed to measure their integrity and detect contaminants that reduce their effectiveness and induce side-effects. Currently, mRNA vaccines and therapies are analysed using a range of time-consuming and costly methods.

Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing.

Library adaptors are short oligonucleotides that are attached to RNA and DNA samples in preparation for next-generation sequencing (NGS). Adaptors can also include additional functional elements, such as sample indexes and unique molecular identifiers, to improve library analysis. Here, we describe Control Library Adaptors, termed CAPTORs, that measure the accuracy and reliability of NGS.

The potential of long noncoding RNA therapies.

The human genome expresses vast numbers of long noncoding RNAs (lncRNA) that fulfil diverse roles in gene regulation, cell biology, development, and human disease. These roles are often mediated by sequence motifs and secondary structures bound by proteins and can regulate epigenetic, transcriptional, and translational pathways. These functional domains can be further optimised and engineered into RNA devices that are widely used in synthetic biology.

Using synthetic chromosome controls to evaluate the sequencing of difficult regions within the human genome.

Background: Next-generation sequencing (NGS) can identify mutations in the human genome that cause disease and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low-complexity, and repetitive regions that are difficult to sequence and analyze. Despite their difficulty, these regions include many clinically important sequences that can inform the treatment of human diseases and improve the diagnostic yield of NGS.

The splicing effect of variants at branchpoint elements in cancer genes.

Purpose: Branchpoint elements are required for intron removal, and variants at these elements can result in aberrant splicing. We aimed to assess the value of branchpoint annotations generated from recent large-scale studies to select branchpoint-abrogating variants, using hereditary cancer genes as model.

Methods: We identified branchpoint elements in 119 genes associated with hereditary cancer from 3 genome-wide experimentally-inferred and 2 predicted branchpoint data sets.

Reporting guidelines for human microbiome research: the STORMS checklist.

The particularly interdisciplinary nature of human microbiome research makes the organization and reporting of results spanning epidemiology, biology, bioinformatics, translational medicine and statistics a challenge. Commonly used reporting guidelines for observational or genetic epidemiology studies lack key features specific to microbiome studies. Therefore, a multidisciplinary group of microbiome epidemiology researchers adapted guidelines for observational and genetic studies to culture-independent human microbiome studies, and also developed new reporting elements for laboratory, bioinformatics and statistical analyses tailored to microbiome studies.

Long-read cDNA sequencing identifies functional pseudogenes in the human transcriptome.

Pseudogenes are gene copies presumed to mainly be functionless relics of evolution due to acquired deleterious mutations or transcriptional silencing. Using deep full-length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we identify here hundreds of novel transcribed pseudogenes expressed in tissue-specific patterns. Some pseudogene transcripts have intact open reading frames and are translated in cultured cells, representing unannotated protein-coding genes.

Testing at scale during the COVID-19 pandemic.

Assembly and publication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome in January 2020 enabled the immediate development of tests to detect the new virus. This began the largest global testing programme in history, in which hundreds of millions of individuals have been tested to date. The unprecedented scale of testing has driven innovation in the strategies, technologies and concepts that govern testing in public health.

Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests.

DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity.

A universal and independent synthetic DNA ladder for the quantitative measurement of genomic features.

Standard units of measurement are required for the quantitative description of nature; however, few standard units have been established for genomics to date. Here, we have developed a synthetic DNA ladder that defines a quantitative standard unit that can measure DNA sequence abundance within a next-generation sequencing library. The ladder can be spiked into a DNA sample, and act as an internal scale that measures quantitative genetics features.

Author Correction: Diagnosis of fusion genes using targeted RNA sequencing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations.

Use of synthetic DNA spike-in controls (sequins) for human genome sequencing.

Next-generation sequencing (NGS) has been widely adopted to identify genetic variants and investigate their association with disease. However, the analysis of sequencing data remains challenging because of the complexity of human genetic variation and confounding errors introduced during library preparation, sequencing and analysis. We have developed a set of synthetic DNA spike-ins-termed 'sequins' (sequencing spike-ins)-that are directly added to DNA samples before library preparation.

Crizotinib and Surgery for Long-Term Disease Control in Children and Adolescents With ALK-Positive Inflammatory Myofibroblastic Tumors.

Purpose: Before anaplastic lymphoma kinase (ALK) inhibitors, treatment options for positive inflammatory myofibroblastic tumors (AP-IMTs) were unsatisfactory. We retrospectively analyzed the outcome of patients with AP-IMT treated with crizotinib to document response, toxicity, survival, and features associated with relapse.

Methods: The cohort comprised eight patients with AP-IMT treated with crizotinib and surgery.

TMPRSS2-ERG fusions linked to prostate cancer racial health disparities: A focus on Africa.

Background: The androgen-regulated gene TMPRSS2 to the ETS transcription factor gene ERG fusion is the most common genomic alteration acquired during prostate tumorigenesis and biased toward men of European ancestry. In contrast, African American men present with more advanced disease, yet their tumors are less likely to acquire TMPRSS2-ERG. Data for Africa is scarce.

Targeted, High-Resolution RNA Sequencing of Non-coding Genomic Regions Associated With Neuropsychiatric Functions.

The human brain is one of the last frontiers of biomedical research. Genome-wide association studies (GWAS) have succeeded in identifying thousands of haplotype blocks associated with a range of neuropsychiatric traits, including disorders such as schizophrenia, Alzheimer's and Parkinson's disease. However, the majority of single nucleotide polymorphisms (SNPs) that mark these haplotype blocks fall within non-coding regions of the genome, hindering their functional validation.

Diagnosis of fusion genes using targeted RNA sequencing.

Fusion genes are a major cause of cancer. Their rapid and accurate diagnosis can inform clinical action, but current molecular diagnostic assays are restricted in resolution and throughput. Here, we show that targeted RNA sequencing (RNAseq) can overcome these limitations.

Chiral DNA sequences as commutable controls for clinical genomics.

Chirality is a property describing any object that is inequivalent to its mirror image. 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.

Synthetic microbe communities provide internal reference standards for metagenome sequencing and analysis.

The complexity of microbial communities, combined with technical biases in next-generation sequencing, pose a challenge to metagenomic analysis. Here, we develop a set of internal DNA standards, termed "sequins" (sequencing spike-ins), that together constitute a synthetic community of artificial microbial genomes. Sequins are added to environmental DNA samples prior to library preparation, and undergo concurrent sequencing with the accompanying sample.

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 have 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.

Machine learning annotation of human branchpoints.

Motivation: The branchpoint element is required for the first lariat-forming reaction in splicing. However current catalogues of human branchpoints remain incomplete due to the difficulty in experimentally identifying these splicing elements. To address this limitation, we have developed a machine-learning algorithm-branchpointer-to identify branchpoint elements solely from gene annotations and genomic sequence.