Multi-omic profiling of pathogen-stimulated primary immune cells.

We performed long-read transcriptome and proteome profiling of pathogen-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors to discover new transcript and protein isoforms expressed during immune responses to diverse pathogens. Long-read transcriptome profiling reveals novel sequences and isoform switching induced upon pathogen stimulation, including transcripts that are difficult to detect using traditional short-read sequencing. Widespread loss of intron retention occurs as a common result of all pathogen stimulations.

Novel gene fusion discovery in Spitz tumours and its relevance in diagnostics.

In addition to morphologic analysis, molecular diagnostic work up of Spitz tumours is often of great value for their accurate diagnosis/classification. Nowadays, next-generation sequencing (NGS) is the predominant screening method in molecular diagnostics. Up to 80% of these melanocytic neoplasms comprise gene fusions as genetic anomalies for which the driver codes for a protein harbouring a kinase domain.

SUsPECT: a pipeline for variant effect prediction based on custom long-read transcriptomes for improved clinical variant annotation.

Our incomplete knowledge of the human transcriptome impairs the detection of disease-causing variants, in particular if they affect transcripts only expressed under certain conditions. These transcripts are often lacking from reference transcript sets, such as Ensembl/GENCODE and RefSeq, and could be relevant for establishing genetic diagnoses. We present SUsPECT (Solving Unsolved Patient Exomes/gEnomes using Custom Transcriptomes), a pipeline based on the Ensembl Variant Effect Predictor (VEP) to predict variant impact on custom transcript sets, such as those generated by long-read RNA-sequencing, for downstream prioritization.

CIRCprimerXL: Convenient and High-Throughput PCR Primer Design for Circular RNA Quantification.

Circular RNA (circRNA) is a class of endogenous non-coding RNA characterized by a back-splice junction (BSJ). In general, large-scale circRNA BSJ detection is performed based on RNA sequencing data, followed by the selection and validation of circRNAs of interest using RT-qPCR with circRNA-specific PCR primers. Such a primer pair is convergent and functional on the circRNA template but divergent and non-functional on the linear host gene.

Performance Evaluation of Three DNA Sample Tracking Tools in a Whole Exome Sequencing Workflow.

Introduction: Next-generation sequencing applications are becoming indispensable for clinical diagnostics. These experiments require numerous wet- and dry-laboratory steps, each one increasing the probability of a sample swap or contamination. Therefore, identity confirmation at the end of the process is recommended to ensure the right data are used for each patient.

Sensitive and Specific Spectral Library Searching with CompOmics Spectral Library Searching Tool and Percolator.

Maintaining high sensitivity while limiting false positives is a key challenge in peptide identification from mass spectrometry data. Here, we investigate the effects of integrating the machine learning-based postprocessor Percolator into our spectral library searching tool COSS (CompOmics Spectral library Searching tool). To evaluate the effects of this postprocessing, we have used 40 data sets from 2 different projects and have searched these against the NIST and MassIVE spectral libraries.

MEIS2 Is an Adrenergic Core Regulatory Transcription Factor Involved in Early Initiation of TH-MYCN-Driven Neuroblastoma Formation.

Roughly half of all high-risk neuroblastoma patients present with MYCN amplification. The molecular consequences of MYCN overexpression in this aggressive pediatric tumor have been studied for decades, but thus far, our understanding of the early initiating steps of MYCN-driven tumor formation is still enigmatic. We performed a detailed transcriptome landscaping during murine TH-MYCN-driven neuroblastoma tumor formation at different time points.

Validation of Circular RNAs Using RT-qPCR After Effective Removal of Linear RNAs by Ribonuclease R.

Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that have been shown to play a role in normal development, homeostasis, and disease, including cancer. CircRNAs are formed through a process called back-splicing, which results in a covalently closed loop with a nonlinear back-spliced junction (BSJ). In general, circRNA BSJs are predicted in RNA sequencing data using one of numerous circRNA detection algorithms.

Personalized Proteome: Comparing Proteogenomics and Open Variant Search Approaches for Single Amino Acid Variant Detection.

Discovery of variant peptides such as a single amino acid variant (SAAV) in shotgun proteomics data is essential for personalized proteomics. Both the resolution of shotgun proteomics methods and the search engines have improved dramatically, allowing for confident identification of SAAV peptides. However, it is not yet known if these methods are truly successful in accurately identifying SAAV peptides without prior genomic information in the search database.

The RNA landscape of the human placenta in health and disease.

The placenta is the interface between mother and fetus and inadequate function contributes to short and long-term ill-health. The placenta is absent from most large-scale RNA-Seq datasets. We therefore analyze long and small RNAs (~101 and 20 million reads per sample respectively) from 302 human placentas, including 94 cases of preeclampsia (PE) and 56 cases of fetal growth restriction (FGR).

COSS: A Fast and User-Friendly Tool for Spectral Library Searching.

Spectral similarity searching to identify peptide-derived MS/MS spectra is a promising technique, and different spectrum similarity search tools have therefore been developed. Each of these tools, however, comes with some limitations, mainly because of low processing speed and issues with handling large databases. Furthermore, the number of spectral data formats supported is typically limited, which also creates a threshold to adoption.

Distinct Notch1 and BCL11B requirements mediate human γδ/αβ T cell development.

γδ and αβ T cells have unique roles in immunity and both originate in the thymus from T-lineage committed precursors through distinct but unclear mechanisms. Here, we show that Notch1 activation is more stringently required for human γδ development compared to αβ-lineage differentiation and performed paired mRNA and miRNA profiling across 11 discrete developmental stages of human T cell development in an effort to identify the potential Notch1 downstream mechanism. Our data suggest that the miR-17-92 cluster is a Notch1 target in immature thymocytes and that miR-17 can restrict BCL11B expression in these Notch-dependent T cell precursors.

SPECS: a non-parametric method to identify tissue-specific molecular features for unbalanced sample groups.

Background: To understand biology and differences among various tissues or cell types, one typically searches for molecular features that display characteristic abundance patterns. Several specificity metrics have been introduced to identify tissue-specific molecular features, but these either require an equal number of replicates per tissue or they can't handle replicates at all.

Results: We describe a non-parametric specificity score that is compatible with unequal sample group sizes.

Closing the circle: current state and perspectives of circular RNA databases.

Circular RNAs (circRNAs) are covalently closed RNA molecules that have been linked to various diseases, including cancer. However, a precise function and working mechanism are lacking for the larger majority. Following many different experimental and computational approaches to identify circRNAs, multiple circRNA databases were developed as well.

High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue specificity.

Transcription initiates at both coding and noncoding genomic elements, including mRNA and long noncoding RNA (lncRNA) core promoters and enhancer RNAs (eRNAs). However, each class has a different expression profile with lncRNAs and eRNAs being the most tissue specific. How these complex differences in expression profiles and tissue specificities are encoded in a single DNA sequence remains unresolved.

Long noncoding RNA expression profiling in cancer: Challenges and opportunities.

In recent years, technological advances in transcriptome profiling revealed that the repertoire of human RNA molecules is more diverse and extended than originally thought. This diversity and complexity mainly derive from a large ensemble of noncoding RNAs. Because of their key roles in cellular processes important for normal development and physiology, disruption of noncoding RNA expression is intrinsically linked to human disease, including cancer.

LNCipedia 5: towards a reference set of human long non-coding RNAs.

While long non-coding RNA (lncRNA) research in the past has primarily focused on the discovery of novel genes, today it has shifted towards functional annotation of this large class of genes. With thousands of lncRNA studies published every year, the current challenge lies in keeping track of which lncRNAs are functionally described. This is further complicated by the fact that lncRNA nomenclature is not straightforward and lncRNA annotation is scattered across different resources with their own quality metrics and definition of a lncRNA.

Expanding the Use of Spectral Libraries in Proteomics.

The 2017 Dagstuhl Seminar on Computational Proteomics provided an opportunity for a broad discussion on the current state and future directions of the generation and use of peptide tandem mass spectrometry spectral libraries. Their use in proteomics is growing slowly, but there are multiple challenges in the field that must be addressed to further increase the adoption of spectral libraries and related techniques. The primary bottlenecks are the paucity of high quality and comprehensive libraries and the general difficulty of adopting spectral library searching into existing workflows.

Targeted Genomic Screen Reveals Focal Long Non-Coding RNA Copy Number Alterations in Cancer Cell Lines.

The landscape of somatic copy-number alterations (SCNAs) affecting long non-coding RNAs (lncRNAs) in human cancers remains largely unexplored. While the majority of lncRNAs remain to be functionally characterized, several have been implicated in cancer development and metastasis. Considering the plethora of lncRNAs genes that have been currently reported, it is conceivable that many more lncRNAs might function as oncogenes or tumor suppressor genes.