Project description:Interventions: Genomic test CANCERPLEX-JP OncoGuide NCC oncopanel system FndationONe CDx genome profile GUARDANT360 MSI Analysis System BRACAnalysis
Primary outcome(s): Development of genome database
Study Design: Single arm Non-randomized
Project description:BmN4 cells are cultured cells derived from Bombyx mori ovaries and widely used to study transposon silencing by PIWI-interacting RNAs (piRNAs). A high-accurate genome sequence of BmN4 cells is required to analyze the piRNA pathway using RNA-seq. The genome sequence of BmN4 cells was assembled using Pacific Biosciences (PacBio) HiFi and Oxford Nanopore technology Ultralong (ONT-UL) reads. Microscopic observation and image analysis showed that BmN4 cells were octoploid on average, and the number of chromosomes per cell was highly variable. We concluded the haplotype-resolved assembly of such a complex genome would be difficult; therefore, we assembled a consensus genome sequence. RNA-seq analysis of Siwi knockdown cells also revealed that Siwi-piRISC may target Countdown (Cd), an LTR retrotransposon. By comparing the consensus genome sequence with the reads, we identified differences between haplotypes, particulary structural variants, suggesting that some transposons, including Countdown, increased their copy number in BmN4 cells.
Project description:The current commonly used single-guide RNA (sgRNA) structure has a shortened duplex compared with the native bacterial clustered regularly interspaced short palindromic repeats RNA (crRNA)–transactivating crRNA (tracrRNA) duplex. Here we show that modifying the sgRNA structure by extending the duplex length and mutating the fourth T of the continuous sequence of Ts (which is the pause signal for RNA polymerase III [pol III]) to C or G significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the new sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss-of-function in non-coding genes.
Project description:Despite the long history of genome assembly research, there remains a large gap between the theoretical and practical work. There is practical software with little theoretical underpinning of accuracy on one hand and theoretical algorithms which have not been adopted in practice on the other. In this paper we attempt to bridge the gap between theory and practice by showing how the theoretical safe-and-complete framework can be integrated into existing assemblers in order to improve contiguity. The optimal algorithm in this framework, called the omnitig algorithm, has not been used in practice due to its complexity and its lack of robustness to real data. Instead, we pursue a simplified notion of omnitigs, giving an efficient algorithm to compute them and demonstrating their safety under certain conditions. We modify two assemblers (wtdbg2 and Flye) by replacing their unitig algorithm with the simple omnitig algorithm. We test our modifications using real HiFi data from the Drosophilia melanogaster and the Caenorhabditis elegans genome. Our modified algorithms lead to a substantial improvement in alignment-based contiguity, with negligible computational costs and either no or a small increase in the number of misassemblies.
Project description:Tunicates are marine invertebrates that compose the closest phylogenetic group to the vertebrates. These chordates present a particularly diverse range of regenerative abilities and life-history strategies. Consequently, tunicates provide an extraordinary perspective into the emergence and diversity of these traits. Here we describe the genome sequencing, annotation and analysis of the Stolidobranchian Botrylloides leachii. We have produced a high-quality 159 Mb assembly, 82% of the predicted 194 Mb genome. Analysing genome size, gene number, repetitive elements, orthologs clustering and gene ontology terms show that B. leachii has a genomic architecture similar to that of most solitary tunicates, while other recently sequenced colonial ascidians have undergone genome expansion. In addition, ortholog clustering has identified groups of candidate genes for the study of colonialism and whole-body regeneration. By analysing the structure and composition of conserved gene linkages, we observed examples of cluster breaks and gene dispersions, suggesting that several lineage-specific genome rearrangements occurred during tunicate evolution. We also found lineage-specific gene gain and loss within conserved cell-signalling pathways. Such examples of genetic changes within conserved cell-signalling pathways commonly associated with regeneration and development that may underlie some of the diverse regenerative abilities observed in tunicates. Overall, these results provide a novel resource for the study of tunicates and of colonial ascidians.
Project description:Genome wide mapping of RNA polymearase III binding sites in Saccharomyces cerevisiae under normal growth and nutrient starved condition using ChIP-seq. Chromatin Immuno-precipitation (ChIP) was performed for FLAG tagged version of pol III subunit RPC128 after crosslinking the log-phase cells with formaldehyde. MOCK and IP DNA was sequenced and coverage of pol III was calculated at each base of the genome.
Project description:With the creation of accurate, chromosome-scale genomes, the next challenge facing the genomics community is the accurate idenfication of transcriptional units, distinguishing them from aberrant transcriptional noise. This has proven to be a challenge as annotation by traditional means, such as short read RNA-seq followed by transcriptome assembly, which is prone to the generation of in-silico artifacts. To address this issue, we took advantage of epigenomic data in the form of ChIP-seq to unbiasedly annotate plant genomes and identify potential annotation issues, as well as identify novel genes. Histone modifications appear in the genome in a reproducible and predictable manner, making them an ideal resource to use in annotation. Trimethylation of histone 3 lysine 4 (H3K4me3), as well as acetylation of histone 3 lysine 56 are well documented to coincide with initiation of transcription by polymerase II (Pol II) at promoter sequences. These initiation marks, paired with marks deposited across the gene body during transcriptional elongation, such as histone 3 lysine 36 tri-methylation (H3K36me3) and histone 3 lysine 4 mono-methylation (H3K4me1), offer a framework to begin identifying complete transcriptional units. We leveraged these data on a genome-wide scale, allowing for identification of annotations discordant with empirical data. In total, 13,159 potential annotation issues were found in Zea mays across three different tissues, which were corroborated using complementary RNA-based approaches. Upon correction and validation, genes were extended by an average of 2,128 base pairs, and the length of discovered novel genes was 1,962 base pairs. Application of this method to five additional plant genomes revealed a variety of novel gene annotations, including 13,836 in Asparagus officianalis, 2,724 in Setaria viridis, 2,446 in Sorghum bicolor, 8,631 in Glycine max, and 2,585 in Phaseolous vulgaris.