Model, Integrate, Search... Repeat: A Sound Approach to Building Integrated Repositories of Genomic Data

A wealth of public data repositories is available to drive genomics and clinical research. However, there is no agreement among the various data formats and models; in the common practice, data sources are accessed one by one, learning their specific descriptions with tedious efforts. In this context, the integration of genomic data and of their describing metadata becomes—at the same time—an important, difficult, and well-recognized challenge. In this chapter, after overviewing the most important human genomic data players, we propose a conceptual model of metadata and an extended architecture for integrating datasets, retrieved from a variety of data sources, based upon a structured transformation process; we then describe a user-friendly search system providing access to the resulting consolidated repository, enriched by a multi-ontology knowledge base. Inspired by our work on genomic data integration, during the COVID-19 pandemic outbreak we successfully re-applied the previously proposed model-build-search paradigm, building on the analogies among the human and viral genomics domains. The availability of conceptual models, related databases, and search systems for both humans and viruses will provide important opportunities for research, especially if virus data will be connected to its host, provider of genomic and phenotype information.

Cohesin modulates DNA replication to preserve genome integrity

Cohesin participates in loop formation by extruding DNA fibers from its ring-shaped structure. Cohesin dysfunction eliminates chromatin loops but only causes modest transcription perturbation, which cannot fully explain the frequently observed mutations of cohesin in various cancers. Here, we found that DNA replication initiates at more than one thousand extra dormant origins after acute depletion of RAD21, a core subunit of cohesin, resulting in earlier replicating timing at approximately 30% of the human genomic regions. In contrast, CTCF is dispensable for suppressing the early firing of dormant origins that are distributed away from the loop boundaries. Furthermore, greatly elevated levels of gross DNA breaks and genome-wide chromosomal translocations arise in RAD21-depleted cells, accompanied by dysregulated replication timing at dozens of hotspot genes. Thus, we conclude that cohesin coordinates DNA replication initiation to ensure proper replication timing and safeguards genome integrity.

Omicron variant of SARS-CoV-2 harbors a unique insertion mutation of putative viral or human genomic origin

The emergence of a heavily mutated SARS-CoV-2 variant (B.1.1.529, Omicron) and it’s spread to 6 continents within a week of initial discovery has set off a global public health alarm. Characterizing the mutational profile of Omicron is necessary to interpret its shared or distinctive clinical phenotypes with other SARS-CoV-2 variants. We compared the mutations of Omicron with prior variants of concern (Alpha, Beta, Gamma, Delta), variants of interest (Lambda, Mu, Eta, Iota and Kappa), and all 1523 SARS-CoV-2 lineages constituting 5.4 million SARS-CoV-2 genomes. Omicron’s Spike protein has 26 amino acid mutations (23 substitutions, two deletions and one insertion) that are distinct compared to other variants of concern. Whereas the substitution and deletion mutations have appeared in previous SARS-CoV-2 lineages, the insertion mutation (ins214EPE) has not been previously observed in any SARS-CoV-2 lineage other than Omicron. The nucleotide sequence encoding for ins214EPE could have been acquired by template switching involving the genomes of other viruses that infect the same host cells as SARS-CoV-2 or the human transcriptome of host cells infected with SARS-CoV-2. For instance, given recent clinical reports of co-infections in COVID-19 patients with seasonal coronaviruses (e.g. HCoV-229E), single cell RNA-sequencing data showing co-expression of the SARS-CoV-2 and HCoV-229E entry receptors (ACE2 and ANPEP) in respiratory and gastrointestinal cells, and HCoV genomes harboring sequences homologous to the nucleotide sequence that encodes ins214EPE, it is plausible that the Omicron insertion could have evolved in a co-infected individual. There is a need to understand the function of the Omicron insertion and whether human host cells are being exploited by SARS-CoV-2 as an ‘evolutionary sandbox’ for host-virus and inter-viral genomic interplay.

Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA

There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types (KRAS and BRAF) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex KRAS G13D (9.82 copies/mL) and 4-plex BRAF V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R2 ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring.

A machine learning case–control classifier for schizophrenia based on DNA methylation in blood

Epigenetic dysregulation is thought to contribute to the etiology of schizophrenia (SZ), but the cell type-specificity of DNA methylation makes population-based epigenetic studies of SZ challenging. To train an SZ case–control classifier based on DNA methylation in blood, therefore, we focused on human genomic regions of systemic interindividual epigenetic variation (CoRSIVs), a subset of which are represented on the Illumina Human Methylation 450K (HM450) array. HM450 DNA methylation data on whole blood of 414 SZ cases and 433 non-psychiatric controls were used as training data for a classification algorithm with built-in feature selection, sparse partial least squares discriminate analysis (SPLS-DA); application of SPLS-DA to HM450 data has not been previously reported. Using the first two SPLS-DA dimensions we calculated a “risk distance” to identify individuals with the highest probability of SZ. The model was then evaluated on an independent HM450 data set on 353 SZ cases and 322 non-psychiatric controls. Our CoRSIV-based model classified 303 individuals as cases with a positive predictive value (PPV) of 80%, far surpassing the performance of a model based on polygenic risk score (PRS). Importantly, risk distance (based on CoRSIV methylation) was not associated with medication use, arguing against reverse causality. Risk distance and PRS were positively correlated (Pearson r = 0.28, P = 1.28 × 10−12), and mediational analysis suggested that genetic effects on SZ are partially mediated by altered methylation at CoRSIVs. Our results indicate two innate dimensions of SZ risk: one based on genetic, and the other on systemic epigenetic variants.

Human sample authentication in biomedical research: comparison of two platforms

Samples used in biomedical research are often collected over years, in some cases from subjects that may have died and thus cannot be retrieved in any way. The value of these samples is priceless. Sample misidentification or mix-up are unfortunately common problems in biomedical research and can eventually result in the publication of incorrect data. Here we have compared the Fluidigm SNPtrace and the Agena iPLEX Sample ID panels for the authentication of human genomic DNA samples. We have tested 14 pure samples and simulated their cross-contamination at different percentages (2%, 5%, 10%, 25% and 50%). For both panels, we report call rate, allele intensity/probability score, performance in distinguishing pure samples and contaminated samples at different percentages, and sex typing. We show that both panels are reliable and efficient methods for sample authentication and we highlight their advantages and disadvantages. We believe that the data provided here is useful for sample authentication especially in biorepositories and core facility settings.

Unbiased sequencing of Mycobacterium tuberculosis urinary cell-free DNA reveals extremely short fragment lengths

Urine cell-free DNA (cfDNA) presents an attractive target for diagnosing pulmonary Mycobacterium tuberculosis (TB) infection but has not been thoroughly characterized. Here, we aimed to investigate the size and composition of TB-derived urine cfDNA with minimal bias using next-generation DNA sequencing (NGS). To enable analysis of highly fragmented urine cfDNA, we used a combination of DNA extraction (Q sepharose) and single-stranded sequence library preparation methods demonstrated to recover short, highly degraded cfDNA fragments. We examined urine cfDNA from ten HIV-positive patients with confirmed pulmonary TB (nine of which had TB cfDNA detectable by qPCR) and two TB-negative controls. TB-derived cfDNA was identifiable by NGS from all TB-positive patients. TB urine cfDNA was significantly shorter than human urine cfDNA, with median fragment lengths of 19–52 bp and 42–92 bp, respectively. TB cfDNA abundance increased exponentially with decreased fragment length, with a peak fragment length of ≤19 bp in most samples. Our methodology also revealed a larger fraction of short human genomic cfDNA than previously reported, with peak fragment lengths of 29–53 bp. Urine cfDNA fragments spanned the TB genome with relative uniformity, but nucleic acids derived from multicopy elements were proportionately overrepresented, providing regions of inherent signal amplification beneficial for molecular diagnosis. This study demonstrates the potential of urine cfDNA as a diagnostic biomarker for TB and will inform improved design of TB urine cfDNA assays. Methods capable of targeting the shortest cfDNA fragments possible will be critical to maximize TB urine cfDNA detection sensitivity.