Project description:Primary objectives: The primary objective is to investigate circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Primary endpoints: circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Project description:Many Cactaceae species exhibit determinate growth of the primary root as a consequence of root apical meristem (RAM) exhaustion. The genetic regulation of this root growth pattern is unknown. We de novo assembled and annotated the root apex transcriptome of the Pachycereus pringlei primary root at three developmental stages with active or exhausted RAM. The assembled transcriptome was characterized and used to evaluate differential transcript expression, identify RT-qPCR reference genes, and infer a transcriptional regulatory network. We generated a robust and comprehensive transcriptome of the primary root apex of P. pringlei and identified putative orthologs of Arabidopsis regulators of RAM maintenance, as well as putative lineage-specific transcripts. Furthermore, the transcriptome contained putative orthologs of most proteins involved in housekeeping processes, hormone signaling, and metabolic pathways. Specific transcriptional programs operate in the root apex at specific developmental time points. The transcriptional state of the P. pringlei root apex as the RAM becomes exhausted is comparable to that of cells from the meristematic, elongation, and differentiation zones of Arabidopsis roots. We suggest that the genetic program underlying the drought stress response is induced during development of the Cactaceae root, and that lineage-specific transcripts could contribute to root apical meristem exhaustion in Cactaceae.
Project description:The naked mole-rat (NMR; Heterocephalus glaber) has recently gained considerable attention in the scientific community for its unique potential to unveil novel insights in the fields of medicine, biochemistry, and evolution. NMRs exhibit unique adaptations that include protracted fertility, cancer resistance, eusociality, and anoxia. This suite of adaptations is not found in other rodent species, suggesting that interrogating conserved and accelerated regions in the NMR genome will find regions of the NMR genome fundamental to their unique adaptations. However, the current NMR genome assembly has limits that make studying structural variations, heterozygosity, and non-coding adaptations challenging. We present a complete diploid naked-mole rat genome assembly by integrating long-read and 10X-linked read genome sequencing of a male NMR and its parents, and Hi-C sequencing in the NMR hypothalamus (N=2). Reads were identified as maternal, paternal or ambiguous (TrioCanu). We then polished genomes with Flye, Racon and Medaka. Assemblies were then scaffolded using the following tools in order: Scaff10X, Salsa2, 3d-DNA, Minimap2-alignment between assemblies, and the Juicebox Assembly Tools. We then subjected the assemblies to another round of polishing, including short-read polishing with Freebayes. We assembled the NMR mitochondrial genome with mitoVGP. Y chromosome contigs were identified by aligning male and female 10X linked reads to the paternal genome and finding male-biased contigs not present in the maternal genome. Contigs were assembled with publicly available male NMR Fibroblast Hi-C-seq data (SRR820318). Both assemblies have their sex chromosome haplotypes merged so that both assemblies have a high-quality X and Y chromosome. Finally, assemblies were evaluated with Quast, BUSCO, and Merqury, which all reported the base-pair quality and contiguity of both assemblies as high-quality. The assembly will next be annotated by Ensembl using public RNA-seq data from multiple tissues (SRP061363). Together, this assembly will provide a high-quality resource to the NMR and comparative genomics communities.
Project description:The naked mole-rat (NMR; Heterocephalus glaber) has recently gained considerable attention in the scientific community for its unique potential to unveil novel insights in the fields of medicine, biochemistry, and evolution. NMRs exhibit unique adaptations that include protracted fertility, cancer resistance, eusociality, and anoxia. This suite of adaptations is not found in other rodent species, suggesting that interrogating conserved and accelerated regions in the NMR genome will find regions of the NMR genome fundamental to their unique adaptations. However, the current NMR genome assembly has limits that make studying structural variations, heterozygosity, and non-coding adaptations challenging. We present a complete diploid naked-mole rat genome assembly by integrating long-read and 10X-linked read genome sequencing of a male NMR and its parents, and Hi-C sequencing in the NMR hypothalamus (N=2). Reads were identified as maternal, paternal or ambiguous (TrioCanu). We then polished genomes with Flye, Racon and Medaka. Assemblies were then scaffolded using the following tools in order: Scaff10X, Salsa2, 3d-DNA, Minimap2-alignment between assemblies, and the Juicebox Assembly Tools. We then subjected the assemblies to another round of polishing, including short-read polishing with Freebayes. We assembled the NMR mitochondrial genome with mitoVGP. Y chromosome contigs were identified by aligning male and female 10X linked reads to the paternal genome and finding male-biased contigs not present in the maternal genome. Contigs were assembled with publicly available male NMR Fibroblast Hi-C-seq data (SRR820318). Both assemblies have their sex chromosome haplotypes merged so that both assemblies have a high-quality X and Y chromosome. Finally, assemblies were evaluated with Quast, BUSCO, and Merqury, which all reported the base-pair quality and contiguity of both assemblies as high-quality. The assembly will next be annotated by Ensembl using public RNA-seq data from multiple tissues (SRP061363). Together, this assembly will provide a high-quality resource to the NMR and comparative genomics communities.
Project description:Porcine 60K BeadChip genotyping arrays (Illumina) are increasingly being applied in pig genomics to validate SNPs identified by re-sequencing or assembly-versus-assembly method. Here we report that more than 98% SNPs identified from the porcine 60K BeadChip genotyping array (Illumina) were consistent with the SNPs identified from the assembly-based method. This result demonstrates that whole-genome de novo assembly is a reliable approach to deriving accurate maps of SNPs.
Project description:We sequenced and analyzed the genome of a highly inbred miniature Chinese pig strain, the Banna Minipig Inbred Line (BMI). we conducted whole genome screening using next generation sequencing (NGS) technology and performed SNP calling using Sus Scrofa genome assembly Sscrofa11.1.
Project description:More than 2x10E9 sequences made on Illumina platform derived from the genome of E14 embryonic stem cells cultured in our laboratory were used to build a database of about 2.7x10E6 single nucleotide variant. The database was validated using other two sequencing datasets from other laboratory and high overlap was observed. The identified variant are enriched on intergenic regions, but several thousands reside on gene exons and regulatory regions, such as promoters, enhancers, splicing site and untranslated regions of RNA, thus indicating high probability of an important functional impact on the molecular biology of this cells. We created a new E14 genome assembly including the new identified variants and used it to map reads from next generation sequencing data generated in our laboratory or in others on E14 cell line. We observed an increase in the number of mapped reads of about 5%. CpG dinucleotide showed the higher variation frequency, probably because of it could be target of DNA methylation. We performed a reduced representation bisulfite sequencing on E14 cell line to test our new genome assembly with respect to the mm9 genome reference. After mapping and methylation status calling, we obtained an increase of about 120,000 called CpG and we avoided about 20,000 wrong CpG calling. genotyping of E14 embryonic stem cells (ESCs) and Reduced representation Bisulfite Sequencing (RRBS) of E14 ESCs.
Project description:In order to polish a long-read genome assembly, short-read illumina data was obtained from Heterodera schachtii cysts (Woensdrecht population from IRS, the Netherlands). Cysts where obtained from infected plant material. Nematodes were cleaned using a sucrose gradient centrifugation step. Thereafter DNA was extracted and used for library preparation and sequencing by Illumina NextSeq500.
Project description:Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromeric sequences are diverse and usually repetitive across species, making them challenging to assemble and identify. Here, we describe centromeres in the model oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus in the nucleus at different life stages and during nuclear division. We report a highly contiguous genome assembly of the P. sojae reference strain, which enabled identification of 15 highly enriched CENP-A binding regions as putative centromeres. By focusing on 10 intact regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the euchromatin mark H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3.