Paulina Galka-Marciniak

Whole-miRNome sequencing: a panel for the targeted sequencing of all human miRNA genes

Abstract Interest in the genetic variation of noncoding genomic elements, including microRNAs (miRNAs), is growing, and several mutations in miRNA genes implicated in human diseases, including cancer, have already been detected. However, the lack of dedicated analytical tools severely hampers progress in this area. In this study, we developed the first whole-miRNome sequencing (WMS) platform, which enables the targeted sequencing of all human miRNA genes (n ∼2000) and 28 miRNA biogenesis genes. By sequencing various types of DNA samples, including ∼300 tumor/normal pairs, from lung, colorectal, ovarian, renal, and basal cell carcinomas, we identified ∼2000 mutations, including 879 in miRNA genes. These mutations were located in all parts of the genes, including seed or cleavage sites essential for the functioning of miRNA genes. The high reliability of the mutations was confirmed through various approaches, including different sequencing methods. The analysis identified several miRNA genes with functional enrichment of cancer mutations, including MIR3928, which was specifically mutated in basal cell carcinoma, suggesting its potential role in this cancer. WMS also allowed the identification of multiple copy number alterations, which often encompassed miRNA genes. WMS provides highly effective, low-cost sequencing of all miRNA genes in different types of samples, including highly degraded ones.

Variant Identification in BARD1 , PRDM9 , RCC1 , and RECQL in Patients with Ovarian Cancer by Targeted Next-generation Sequencing of DNA Pools

Abstract Several ovarian cancer susceptibility genes have been discovered, but more are likely to exist. In this study, we aimed to analyze knowledge-based selected genes, that is, BARD1, PRDM9, RCC1, and RECQL, in which pathogenic germline variants have been reported in patients with breast and/or ovarian cancer. As deep sequencing of DNA samples remains costly, targeted next-generation sequencing of DNA pools was utilized to screen the exons of BARD1, PRDM9, RCC1, and RECQL in approximately 400 Polish ovarian cancer cases. A total of 25 pools of 16 samples (including several duplicated samples with known variants) were sequenced on the NovaSeq6000 and analyzed with SureCall (Agilent) application. The set of variants was filtrated to exclude spurious variants, and, subsequently, the identified rare genetic variants were validated using Sanger sequencing. No pathogenic mutation was found within the analyzed cohort of patients with ovarian cancer. Validation genotyping of filtered rare silent and missense variants revealed that the majority of them were true alterations, especially those with a higher mutation quality value. The high concordance (R2 = 0.95) of population allele frequency for 44 common SNPs in the European control population (gnomAD) and our experiment confirmed the reliability of pooled sequencing. Mutations in BARD1, PRDM9, RCC1, and RECQL do not contribute substantially to the risk of ovarian cancer. Pooled DNA sequencing is a cost-effective and reliable method for the initial screening of candidate genes; however, it still requires validation of identified rare variants. Prevention Relevance: BARD1, PRDM9, RCC1, and RECQL are not high/moderate-risk ovarian cancer susceptibility genes. Pooled sequencing is a reliable and cost-effective method to detect rare variants in candidate genes.