Biology subjects

Kalinava, N.

Publications and source records attributed to Kalinava, N..

3 recordsLinked to original sources

bioRxiv2020

Mis-annotated multi nucleotide variants in public cancer genomics datasets can lead to inaccurate mutation calls with significant implications

BackgroundNext generation sequencing is widely used in cancer to profile tumors and detect variants. Most somatic variant callers used in these pipelines identify variants at the lowest possible granularity - single nucleotide variants (SNVs). As a result, multiple adjacent SNVs are called individually instead of as a multi-nucleotide variant (MNV). The problem with this level of granularity is that the amino acid change from the individual SNVs within a codon could be different from the amino acid change based on the MNV that results from combining the SNVs. Most variant annotation tools do not account for this, leading to incorrect conclusions about the downstream effects of the variants. MethodHere, we used Variant Call Files (VCFs) from the TCGA Mutect2 caller, and developed a solution to merge SNVs to MNVs. Our custom script takes the phasing information from the SNV VCFs and based on a gene model, determines if SNVs are at the same codon and need to be merged into a MNV prior to variant annotation. ResultsWe analyzed 10,383 VCFs from TCGA and found 12,141 MNVs that were incorrectly annotated. Strikingly, the analysis of seven commonly mutated genes from 178 studies from cBioPortal revealed that MNVs were consistently missed in 20 of these studies, while they were correctly annotated in 15 more recent studies. The best and most common example of MNVs was found at the BRAF V600 locus, where several public datasets reported separate BRAF V600E and BRAF V600M variants, instead of a single merged V600K variant. ConclusionWhile some datasets merged MNVs correctly, many public datasets have not been corrected for this problem. As a best practice for variant calling, we recommend that MNVs be accounted for in NGS processing pipelines, thus improving analyses on the impact of somatic variants in cancer genomics.

genomics↗

bioRxiv2018

Developmentally regulated transcription and silencing cycle of nuclear RNAi-targeted LTR retrotransposons in Caenorhabditis elegans

Small RNA-guided chromatin silencing, also referred to as nuclear RNAi, plays an essential role in genome surveillance in eukaryotes and provides a unique paradigm to explore the complexity in RNA-mediated chromatin regulation and transgenerational epigenetics. A well-recognized paradox in this research area is that transcription of the target loci is necessary for the initiation and maintenance of the silencing at the same loci. How the two opposing activities (transcriptional activation and repression) are coordinated during animal development is poorly understood. To resolve this gap, we took single-molecule RNA imaging, deep-sequencing, and genetic approaches towards delineating the developmental regulation and subcellular localization of RNA transcripts of two exemplary endogenous germline nuclear RNAi targets in C. elegans, Cer3 and Cer8 LTR retrotransposons. By examining the wild type and a collection of mutant strains, we found that transcription and silencing cycle of Cer3 and Cer8 is tightly coupled with the early embryogenesis and germline mitotic and meiotic cell cycles. Strikingly, Cer3 and Cer8 transcripts are exclusively localized in the nuclei of germ cells in both wild type and germline nuclear RNAi-defective mutant animals. RNA-sequencing analysis found that this nuclear enrichment feature is a general feature for the endogenous targets of the germline nuclear RNAi pathway. In addition, the germline and somatic repressions of Cer3 have different genetic requirement for the three H3K9 histone methyltransferases, MET-2, SET-25, and SET-32, in conjunction with the nuclear Argonaute protein WAGO-9/HRDE-1. These results provide a first comprehensive cellular and developmental characterization of the nuclear RNAi-targeted endogenous targets throughout animal reproductive cycle. Altogether, these results support a model in which (1) both the transcriptional activation and repression steps of the germline nuclear RNAi pathway are tightly coupled with animal development, (2) the endogenous targets exhibit a hallmark of nuclear enrichment of their transcripts, and (3) different heterochromatin enzymes play distinct roles in somatic and germline silencing of the endogenous targets.

developmental biology↗

bioRxiv2018

Caenorhabditis elegans heterochromatin factor SET-32 plays an essential role in transgenerational establishment of nuclear RNAi-mediated epigenetic silencing

Epigenetic inheritance contributes fundamentally to transgenerational physiology and fitness. Mechanistic understanding of RNA-mediated chromatin modification and transgenerational epigenetic inheritance, which in C. elegans can be triggered by exogenous double-stranded RNA (exo-dsRNA) or facilitated by endogenous small interfering RNAs (endo-siRNAs), has mainly been limited to the post-initiation phases of silencing. Indeed, the dynamic process by which nuclear RNAi engages a transcriptionally active target, before the repressive state is stably established, remains largely a mystery. Here we found that the onset of exo-dsRNA-induced nuclear RNAi is a transgenerational process, and that establishment requires SET-32, one of the three putative histone methyltransferases (HMTs) that are required for H3K9me3 deposition at the nuclear RNAi targets. We also performed multigenerational whole-genome analyses to examine the establishment of silencing at endogenous targets of germline nuclear RNAi. The nuclear Argonaute protein HRDE-1 is essential for the maintenance of nuclear RNAi. Repairing a loss-of-function mutation in hrde-1 by CRISPR restored the silencing of endogenous targets in animals carrying wild type set-32. However, for numerous endogenous targets, repairing the hrde-1 mutation in a set-32;hrde-1 double mutant failed to restore their silencing states in up to 20 generations after the hrde-1 repair, using a similar genome editing approach. We found that despite a prominent role in the establishment of silencing, however, set-32 is completely dispensable for the maintenance of silencing once HRDE-1-dependent gene repression is established. Our study indicates that: 1) establishment and maintenance of siRNA-guided transcriptional repression are two distinct processes with different genetic requirements; and 2) the rate-limiting step of the establishment phase is a transgenerational, chromatin-based process. In addition, our study reveals a novel paradigm in which a heterochromatin factor primarily functions to promote the establishment of transgenerational silencing, expanding mechanistic understanding of the well-recognized role of heterochromatin in epigenetic maintenance.

molecular biology↗