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:We applied the RNA-Seq approach to reconstruct the transcriptome of Vitis vinifera cv. Corvina, using RNA pooled from a comprehensive set of sampled tissues in different organs and development steps, and we were able to reconstruct some novel and putative private Corvina genes. We analyzed the expression of these genes in three berry developmental conditions, and posit that they may play some role in the formation of the mature organ. Background: Plants display a high genetic and phenotypic variability among different cultivars. Understanding the genetic components that contribute to phenotypic diversity is necessary to disentangle genetic factors from the environment. Given the high degree of genetic diversity among plant cultivars a whole-genome sequencing and re-annotation of each variety is required but a reliable genome assembly is hindered by the high heterozigosity and sequence divergence. Results: we show the feasibility of an approach based on sequencing of cDNA by RNA-Seq to analyze varietal diversity between a local grape cultivar Corvina and the PN40024 grape reference genome. We detected 15,260 known genes and we annotated alternative splicing isoforms for 9,463 genes. Our approach allowed to define 2,321 protein coding putative novel genes in unannotated or unassembled regions of the reference genome PN40024 and 180 putative private Corvina genes whose sequence is not shared with the reference genome. Conclusions: With a de novo assembly based approach we were able to reconstruct a substantial part of the Corvina transcriptome and we improved substantially known genes annotations by better defining the structure of known genes, annotating splicing isoforms and detecting unannotated genes. Moreover our results clearly define sets of private genes which are likely part of the âdispensableâ genome and potentially involved into influencing some cultivar-specific characteristics. In plant biology a transcriptome de novo assembly approach should not be limited to species where no reference genome is available as it can improve the annotation lead to the identification of genes peculiar of a cultivar.
Project description:The genus Abiotrophia represents a heterogeneous group of fastidious cocci that show a dependence on pyridoxal hydrochloride analogs for growth. The genetic heterogeneity in the genus Abiotrophia was examined by DNA-DNA hybridization, PCR assay of genomic DNA sequences, and restriction fragment length polymorphism and sequence homology analyses of the PCR-amplified 16S rRNA gene. Nine type or reference strains of Abiotrophia defectiva, Abiotrophia adiacens, and Abiotrophia elegans and 36 oral Abiotrophia isolates including the ones presumptively identified as Gemella morbillorum by the rapid ID32 STREP system were divided into four groups: A. defectiva (genotype 1), A. adiacens (genotype 2), A. elegans (genotype 4), and a fourth species (genotype 3) which we propose be named Abiotrophia para-adiacens sp. nov. A PCR assay specific for detection and identification of the novel Abiotrophia species was developed. A. para-adiacens generally produced beta-glucosidase but did not produce alpha- or beta-galactosidase or arginine dihydrolase, did not ferment, trehalose, pullulan, or tagatose, and was serotype IV, V, or VI. Thus, it was distinguished phenotypically from A. adiacens, A. elegans, and A. defectiva as well as, apparently, from the recently described species Abiotrophia balaenopterae sp. nov., which produces arginine dihydrolase and which ferments pullulan but not sucrose (P. A. Lawson et al., Int. J. Syst. Bacteriol. 49:503-506, 1999). Strain ATCC 27527, currently listed as G. morbillorum, was a member of the species A. para-adiacens.
Project description:For this project, we have sequenced, assembled and annotated a transcriptome of a diploid wheat Triticum urartu accession PI 428198. The sequencing libraries were prepared from shoot and root tissues harvested from 2-3 week old seedlings. All sequencing was carried out on the Illumina HiSeq platform using the 100 bp pair-end protocol (248.5 million reads). The assembly was constructed using a multiple k-mer approach with a de novo assembly algorithm implemented in CLC Genomics Workbench 5.5 and additional redundancy reduction with CD-HIT and blast2cap3 programs. Open reading frames and proteins were predicted using BLASTX searches and a findorf algorithm.
Project description:Abiotrophia adiacens and Abiotrophia defectiva, previously referred to as nutritionally variant streptococci, Streptococcus adjacens and Streptococcus defectivus, respectively, are causes of infective endocarditis. We describe a method of identifying these two species and also of distinguishing them from 15 other major etiological pathogens of infective endocarditis by means of 16S rRNA gene PCR amplification followed by restriction fragment length polymorphism analysis (PCR-RFLP). The 16S rRNA genes were successfully amplified with a set of universal primers from all 17 species of bacteria examined, including viridans group streptococci. The RFLP patterns of A. adiacens and A. defectiva obtained by HaeIII or MspI digestion were readily distinguished from each other and from those of other bacteria. When PCR analysis was performed with the supernatant of a suspension of a boiled colony, the 16S rRNA genes of 80 of 82 isolates (97%) of A. adiacens and all isolates (11 of 11) of A. defectiva were amplified. The HaeIII RFLP patterns of the isolates were the same as those of the corresponding type strains, although 28% of A. adiacens isolates revealed intraspecies polymorphism. The detection limit of this method was 0.1 pg of genomic DNA, as assessed by using the digoxigenin-labeling DNA detection system. Thus, the PCR-RFLP analysis that we developed is applicable for the routine detection of Abiotrophia from clinical specimens.
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.