Biology subjects

Bucris, E.

Publications and source records attributed to Bucris, E..

3 recordsLinked to original sources

bioRxiv2024

Novel Amplicons based Method for Near-Full-Length Genome (NFLG) Sequencing of HIV-1 Group M Recombinant Forms

Over the years the spread of HIV-1 across the globe resulted in the creation of multiple subtypes and new recombinant forms (RFs). While the pol gene region of the HIV-1 genome is used for resistance mutations analysis and initial detection of RFs, whole genome sequencing analysis is required to determine recombination events across the viral genome. Here, we present a novel robust near-full length genome (NFLG) sequencing approach for the sequencing of HIV-1 genomes, out of clinical whole blood samples. This method has been successfully tested for various HIV-1 subtypes and RFs. The method is based on an in-house developed set of 32 pan-genotypic primer pairs, divided into two pools, each containing 16 primer pairs covering the entire HIV-1 genome. Two parallel multiplex PCR reactions were used to generate 32 overlapping DNA fragments spanning the HIV-1 genome. Nextera XT protocol was used to obtain barcoded DNA libraries, which were sequenced with the Illumina Miseq platform using a V3 kit. A consensus sequence was determined for each sample and was used to define recombination events across the genome. For this aim, a combined analysis of several computational tools including HIV BLAST, phylogenetic analysis, RIP, SimPlot++ and jpHMM were employed. Overall, plasma samples from 33 patients suspected to carry RFs and 2 different, known pure subtypes controls, were included in this study. Genome coverage varied between RFs, while the gag and pol genes were nearly fully covered, the highly variable env gene region was not. Yet, these NFLG analyses enabled the identification of recombination events genome wide. In summary, we describe a methodology for HIV-1 NFLG sequencing, which is based on partially overlapping, multiple PCR fragments, spanning the HIV-1 genome. Additionally, this newly refined method was compared to HIV-1 NFLG results of PCR-free metagenomic sequencing and proved to obtain greater coverage of the HXB2 reference genome. Yet, further testing and validation on a larger cohort is required. Still, this method enables sequencing of 20 different patient samples in a single MiSeq sequencing run and was used for the characterization of different HIV-1 RFs and pure subtypes circulating in Israel.

molecular biology↗

bioRxiv2019

Characterization of splice isoform switching during human kidney development

Nephrons are the functional units of the kidney. During kidney development, cells from the cap mesenchyme - a transient kidney-specific progenitor state - undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron. Faults in this transition can lead to a pediatric malignancy of the kidney called Wilms tumor that mimics normal kidney development. While kidney development has been characterized at the gene expression level, a comprehensive characterization of alternative splicing is lacking. We therefore performed RNA sequencing on cell populations representing early, intermediate, and late developmental stages of the human fetal kidney, as well as three blastemal-predominant Wilms tumor patient-derived xenografts. We identified a set of transcripts that are alternatively spliced between the different developmental stages. Moreover, we found that cells from the earliest developmental stage have a mesenchymal splice-isoform profile that is similar to that of blastemal-predominant Wilms tumors. RNA binding motif enrichment analysis suggests that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate mRNA splice isoform switching during human kidney development. These findings illuminate new molecular mechanisms involved in kidney development and pediatric kidney tumors.\n\nHIGHLIGHTSO_LIDuring fetal kidney development, kidney progenitor cells undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron.\nC_LIO_LIRNA sequencing identifies a set of transcripts that undergo splice isoform switching during the mesenchymal to epithelial transition (MET) that occurs in the course of human fetal kidney development.\nC_LIO_LICells in the early stages of kidney development have a mesenchymal splice-isoform profile that is similar to that observed in blastemal-predominant Wilms tumor patient-derived xenografts (WT-PDX) that represent an aggressive subtype of Wilms tumors.\nC_LIO_LIRNA binding motif enrichment analysis indicates that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate splice isoform switching during human kidney development.\nC_LI

genomics↗

bioRxiv2019

Single-cell RNA sequencing reveals mRNA splice isoform switching during kidney development

During mammalian kidney development, nephron progenitors undergo a mesenchymal to epithelial transition and eventually differentiate into the various tubular segments of the nephron. Recently, the different cell types in the developing kidney were characterized using the Dropseq single cell RNA sequencing technology for measuring gene expression from thousands of individual cells. However, many genes can also be alternatively spliced and this creates an additional layer of heterogeneity. We therefore used full transcript length single-cell RNA sequencing to obtain the transcriptomes of 544 individual cells from mouse embryonic kidneys. We first used gene expression levels to identify each cell type. Then, we comprehensively characterized the splice isoform switching that occurs during the transition between mesenchymal and epithelial cellular states and identified several putative splicing regulators, including the genes Esrp1/2 and Rbfox1/2. We anticipate that these results will improve our understanding of the molecular mechanisms involved in kidney development.

genomics↗