Evaluation of Tn5 protein fusions suitable for GET-seq
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ABSTRACT:
PROVIDER: PRJEB40722 | ENA |
REPOSITORIES: ENA
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Project description:Tn5 transposase is used as a tool for detecting nucleosome-free regions of genomic DNA in eukaryotes, but its DNA target site in chromatin has not been understood. In the present study, the well-positioned dinucleosomes were reconstituted, and the Tn5 transposase target sites were mapped in the dinucleosomes in vitro. We found that Tn5 transposase preferentially targets near the entry-exit DNA region within the nucleosome, if the linker DNA exists between two nucleosomes. This specific DNA targeting by Tn5 did not depend on the linker DNA length and DNA sequence. Tn5 transposase becomes to target the middle of the linker DNA, in addition to the entry-exit site of the nucleosome, if the linker DNA length extends to 30 base pairs. These in vitro data provide direct evidence for the Tn5 target sites in the nucleosome, resulting important information for interpretation of the Tn5-transposase-based genomics methods, which have been interpreted as linker or nucleosome-free DNA regions in genomes.
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Project description:Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from sub-picogram amounts of cDNA. Comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, as naked Tn5 can be annealed to any oligonucleotide of choice, for example molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enables innovation in sequencing-based applications.
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Project description:Tn5 transposon tagments double-strand DNA and RNA/DNA hybrid to generate nucleic acids ready to be amplified for high throughput sequencing. The nucleic acid substrates of Tn5 transposon need to be explored to increase the applications of Tn5. Here, we find that Tn5 transposon is able to transport oligos into the 5’ end of single-strand DNA, which are more than 140 base pairs of nucleic acids. Based on this property of Tn5, we develop a tagmentation-based and ligation-enabled single-strand DNA sequencing method, TABLE-seq. Through a series of reaction temperature, time, and enzyme concentration tests, we managed to apply this TABLE-seq to strand-specific RNA sequencing, starting with as low as 30 picograms of total RNA. Moreover, compared with traditional dUTP based strand-specific RNA sequencing, this method detects more genes, has higher strand-specificity, and shows more evenly distributed reads across genes. Together, our results provide insights into the properties of Tn5 transposons and expand the applications of Tn5 in cutting-edge sequencing techniques.
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Project description:Fusion genes can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. The BRAF gene is frequently involved in oncogenic fusions, with fusion frequencies of 0.2-3% throughout different cancers. However, BRAF fusions rarely occur in the same gene configuration, potentially challenging personalized therapy design. In particular, the influence that is imposed by the wide variety of fusion partners on the oncogenic role of BRAF during tumor growth and drug response is unknown. Here, we used patient-derived colorectal cancer organoids to functionally characterize and cross-compare previously identified BRAF fusions containing various partner genes (AGAP3, DLG1 and TRIM24) with respect to cellular behaviour, downstream signaling activation and response to targeted therapies. We demonstrate that 5’ partner choice of BRAF fusions affects their subcellular localization and intracellular signaling capacity. In particular the DLG1-BRAF fusion protein showed distinct localization to the plasma membrane and exhibited increased activation of downstream MAPK signaling under unperturbed conditions. Moreover, phosphoproteomics and RNA sequencing identified distinct subsets of affected signaling pathways and altered gene expression of BRAF fusions. The different BRAF fusions exhibited varying sensitivities to simultaneous targeted inhibition of MEK and the EGF receptor family. However, all BRAF fusions conveyed resistance to targeted monotherapy against the EGF receptor family, suggesting that BRAF fusions should be screened alongside other MAPK pathway alterations to identify mCRC patients to exclude from cetuximab treatment
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Project description:The analysis of chromatin features in single cells centers around the use of Tn5 transposase and exploits its activity to simultaneously fragment target DNA and integrate adapter sequences of choice. This reaction provides a direct readout in the transposase-accessible chromatin in single cells (scATAC-seq) assay to map open chromatin regions. Furthermore, by targeting Tn5 to antibody-bound chromatin epitopes, features like histone modifications can be mapped in single cells. Thus, enhancing Tn5 activity to improve genomic coverage for scATAC-seq or facilitating multi-omics readout of chromatin features via Tn5 together with the transcriptome is of great interest. Here, we address these issues by optimizing scATAC-seq for an increased number of integrations per cell. In addition, we provide a protocol that combines mapping of histone modification with scRNA-seq from the same cell. Our experimental workflows improve the results obtained from the downstream data analysis and serve to better resolve epigenetic heterogeneity and transcription regulation in single cells.
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