A bumpy ride on the diagnostic bench of massive parallel sequencing, the case of the mitochondrial genome

The advent of massive parallel sequencing (MPS) has revolutionized the field of human molecular genetics, including the diagnostic study of mitochondrial (mt) DNA dysfunction. The analysis of the complete mitochondrial genome using MPS platforms is now common and will soon outrun conventional sequencing. However, the development of a robust and reliable protocol is rather challenging. A previous pilot study for the re-sequencing of human mtDNA revealed an uneven coverage, affecting predominantly part of the plus strand. In an attempt to address this problem, we undertook a comparative study of standard and modified protocols for the Ion Torrent PGM system. We could not improve strand representation by altering the recommended shearing methodology of the standard workflow or omitting the DNA polymerase amplification step from the library construction process. However, we were able to associate coverage bias of the plus strand with a specific sequence motif. Additionally, we compared coverage and variant calling across technologies. The same samples were also sequenced on a MiSeq device which showed that coverage and heteroplasmic variant calling were much improved

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Accurate and comprehensive analysis of single nucleotide variants and large deletions of the human mitochondrial genome in DNA and single cells

Massive parallel sequencing (MPS) can accurately quantify mitochondrial DNA (mtDNA) single nucleotide variants (SNVs), but no MPS methods are currently validated to simultaneously and accurately establish the breakpoints and frequency of large deletions at low heteroplasmic loads. Here we present the thorough validation of an MPS protocol to quantify the load of very low frequency, large mtDNA deletions in bulk DNA and single cells, along with SNV calling by standard methods. We used a set of well-characterized DNA samples, DNA mixes and single cells to thoroughly control the study. We developed a custom script for the detection of mtDNA rearrangements that proved to be more accurate in detecting and quantifying deletions than pre-existing tools. We also show that PCR conditions and primersets must be carefully chosen to avoid biases in the retrieved variants and an increase in background noise, and established a lower detection limit of 0.5% heteroplasmic load for large deletions, and 1.5 and 2% for SNVs, for bulk DNA and single cells, respectively. Finally, the analysis of different single cells provided novel insights into mtDNA cellular mosaicism.European Journal of Human Genetics advance online publication, 23 August 2017; doi:10.1038/ejhg.2017.129.status: publishe

Liquid Biopsy-Derived DNA Sources as Tools for Comprehensive Mutation Profiling in Multiple Myeloma: A Comparative Study

The analysis of bone marrow (BM) samples in multiple myeloma (MM) patients can lead to the underestimation of the genetic heterogeneity within the tumor. Blood-derived liquid biopsies may provide a more comprehensive approach to genetic characterization. However, no thorough comparison between the currently available circulating biomarkers as tools for mutation profiling in MM has been published yet and the use of extracellular vesicle-derived DNA for this purpose in MM has not yet been investigated. Therefore, we collected BM aspirates and blood samples in 30 patients with active MM to isolate five different DNA types, i.e., cfDNA, EV-DNA, BM-DNA and DNA isolated from peripheral blood mononucleated cells (PBMNCs-DNA) and circulating tumor cells (CTC-DNA). DNA was analyzed for genetic variants with targeted gene sequencing using a 165-gene panel. After data filtering, 87 somatic and 39 germline variants were detected among the 149 DNA samples used for sequencing. cfDNA showed the highest concordance with the mutation profile observed in BM-DNA and outperformed EV-DNA, CTC-DNA and PBMNCs-DNA. Of note, 16% of all the somatic variants were only detectable in circulating biomarkers. Based on our analysis, cfDNA is the preferable circulating biomarker for genetic characterization in MM and its combined use with BM-DNA allows for comprehensive mutation profiling in MM

Liquid Biopsy-Derived DNA Sources as Tools for Comprehensive Mutation Profiling in Multiple Myeloma: A Comparative Study

The analysis of bone marrow (BM) samples in multiple myeloma (MM) patients can lead to the underestimation of the genetic heterogeneity within the tumor. Blood-derived liquid biopsies may provide a more comprehensive approach to genetic characterization. However, no thorough comparison between the currently available circulating biomarkers as tools for mutation profiling in MM has been published yet and the use of extracellular vesicle-derived DNA for this purpose in MM has not yet been investigated. Therefore, we collected BM aspirates and blood samples in 30 patients with active MM to isolate five different DNA types, i.e. cfDNA, EV-DNA, BM-DNA and DNA isolated from peripheral blood mononucleated cells (PBMNCs-DNA) and circulating tumor cells (CTC-DNA). DNA was analyzed for genetic variants with targeted gene sequencing using a 165-gene panel. After data filtering, 87 somatic and 39 germline variants were detected among the 149 DNA samples used for sequencing. cfDNA showed the highest concordance with the mutation profile observed in BM-DNA and outperformed EV-DNA, CTC-DNA and PBMNCs-DNA. Of note, 16% of all the somatic variants were only detectable in circulating biomarkers. Based on our analysis, cfDNA is the preferable circulating biomarker for genetic characterization in MM and its combined use with BM-DNA allows for comprehensive mutation profiling in MM.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Identification of candidate cancer predisposing variants by performing whole-exome sequencing on index patients from BRCA1 and BRCA2-negative breast cancer families

Abstract Background In the majority of familial breast cancer (BC) families, the etiology of the disease remains unresolved. To identify missing BC heritability resulting from relatively rare variants (minor allele frequency ≤ 1%), we have performed whole exome sequencing followed by variant analysis in a virtual panel of 492 cancer-associated genes on BC patients from BRCA1 and BRCA2 negative families with elevated BC risk. Methods BC patients from 54 BRCA1 and BRCA2-negative families with elevated BC risk and 120 matched controls were considered for germline DNA whole exome sequencing. Rare variants identified in the exome and in a virtual panel of cancer-associated genes [492 genes associated with different types of (hereditary) cancer] were compared between BC patients and controls. Nonsense, frame-shift indels and splice-site variants (strong protein-damaging variants, called PDAVs later on) observed in BC patients within the genes of the panel, which we estimated to possess the highest probability to predispose to BC, were further validated using an alternative sequencing procedure. Results Exome- and cancer-associated gene panel-wide variant analysis show that there is no significant difference in the average number of rare variants found in BC patients compared to controls. However, the genes in the cancer-associated gene panel with nonsense variants were more than two-fold over-represented in women with BC and commonly involved in the DNA double-strand break repair process. Approximately 44% (24 of 54) of BC patients harbored 31 PDAVs, of which 11 were novel. These variants were found in genes associated with known or suspected BC predisposition (PALB2, BARD1, CHEK2, RAD51C and FANCA) or in predisposing genes linked to other cancer types but not well-studied in the context of familial BC (EXO1, RECQL4, CCNH, MUS81, TDP1, DCLRE1A, DCLRE1C, PDE11A and RINT1) and genes associated with different hereditary syndromes but not yet clearly associated with familial cancer syndromes (ABCC11, BBS10, CD96, CYP1A1, DHCR7, DNAH11, ESCO2, FLT4, HPS6, MYH8, NME8 and TTC8). Exome-wide, only a few genes appeared to be enriched for PDAVs in the familial BC patients compared to controls. Conclusions We have identified a series of novel candidate BC predisposition variants/genes. These variants/genes should be further investigated in larger cohorts/case-control studies. Other studies including co-segregation analyses in affected families, locus-specific loss of heterozygosity and functional studies should shed further light on their relevance for BC risk

Healthcare-Associated COVID-19 across Five Pandemic Waves: Prediction Models and Genomic Analyses

Background: Healthcare-associated SARS-CoV-2 infections need to be explored further. Our study is an analysis of hospital-acquired infections (HAIs) and ambulatory healthcare workers (aHCWs) with SARS-CoV-2 across the pandemic in a Belgian university hospital. Methods: We compared HAIs with community-associated infections (CAIs) to identify the factors associated with having an HAI. We then performed a genomic cluster analysis of HAIs and aHCWs. We used this alongside the European Centre for Disease Control (ECDC) case source classifications of an HAI. Results: Between March 2020 and March 2022, 269 patients had an HAI. A lower BMI, a worse frailty index, lower C-reactive protein (CRP), and a higher thrombocyte count as well as death and length of stay were significantly associated with having an HAI. Using those variables to predict HAIs versus CAIs, we obtained a positive predictive value (PPV) of 83.6% and a negative predictive value (NPV) of 82.2%; the area under the ROC was 0.89. Genomic cluster analyses and representations on epicurves and minimal spanning trees delivered further insights into HAI dynamics across different pandemic waves. The genomic data were also compared with the clinical ECDC definitions for HAIs; we found that 90.0% of the ‘definite’, 87.8% of the ‘probable’, and 70.3% of the ‘indeterminate’ HAIs belonged to one of the twenty-two COVID-19 genomic clusters we identified. Conclusions: We propose a novel prediction model for HAIs. In addition, we show that the management of nosocomial outbreaks will benefit from genome sequencing analyses

Intracerebral administration of CTLA-4 and PD-1 immune checkpoint blocking monoclonal antibodies in patients with recurrent glioblastoma: A phase i clinical trial

Background Patients with recurrent glioblastoma (rGB) have a poor prognosis with a median overall survival (OS) of 30-39 weeks in prospective clinical trials. Intravenous administration of programmed cell death protein 1 and cytotoxic T-lymphocyte-associated antigen 4 inhibitors has low activity in patients with rGB. In this phase I clinical trial, intracerebral (IC) administration of ipilimumab (IPI) and nivolumab (NIVO) in combination with intravenous administration of NIVO was investigated. Methods Within 24 hours following the intravenous administration of a fixed dose (10 mg) of NIVO, patients underwent a maximal safe resection, followed by injection of IPI (10 mg; cohort-1), or IPI (5 mg) plus NIVO (10 mg; cohort-2) in the brain tissue lining the resection cavity. Intravenous administration of NIVO (10 mg) was repeated every 2 weeks (max. five administrations). Next generation sequencing and RNA gene expression profiling was performed on resected tumor tissue. Results Twenty-seven patients were enrolled (cohort-1: n=3; cohort-2: n=24). All patients underwent maximal safe resection and planned IC administrations and preoperative NIVO. Thirteen patients (cohort-1: n=3; cohort-2: n=10) received all five postoperative intravenous doses of NIVO. In cohort-2, 14 patients received a median of 3 (range 1-4) intravenous doses. Subacute postoperative neurological deterioration (n=2) was reversible on steroid treatment; no other central nervous system toxicity was observed. Immune-related adverse events were infrequent and mild. GB recurrence was diagnosed in 26 patients (median progression-free survival (PFS) is 11.7 weeks (range 2-152)); 21 patients have died due to progression. Median OS is 38 weeks (95% CI: 27 to 49) with a 6-month, 1-year, and 2-year OS-rate of, respectively, 74.1% (95% CI: 57 to 90), 40.7% (95% CI: 22 to 59), and 27% (95% CI: 9 to 44). OS compares favorable against a historical cohort (descriptive Log-Rank p>0.003). No significant difference was found with respect to PFS (descriptive Log-Rank test p>0.05). A higher tumor mRNA expression level of B7-H3 was associated with a significantly worse survival (multivariate Cox logistic regression, p>0.029). Conclusion IC administration of NIVO and IPI following maximal safe resection of rGB was feasible, safe, and associated with encouraging OS. Trial registration NCT03233152.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Nucleotide GC, AC and CT bias plots for the human mtDNA.

<p>The relative coverage as seen in this illustration is based on the average of the relative coverage of the six samples processed with the different protocols: Covaris shearing followed by the Ion Torrent protocol, Covaris shearing followed by the TruSeq procedure and the Nextera XT method. The average relative coverage was calculated for the total relative coverage and for both strand separately.</p

Overview of the minor allele frequency (in %) of heteroplasmic variants detected in this study.

<p>Overview of the minor allele frequency (in %) of heteroplasmic variants detected in this study.</p