Project description:Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. However, all TIS methods described to date use short nucleotide sequence reads which cannot uniquely determine the locations of transposon insertions within repeating genomic sequences where the repeat units are longer than the sequence read length. To overcome this limitation, we have developed a TIS method using Oxford Nanopore sequencing technology that generates and uses long nucleotide sequence reads; we have called this method LoRTIS (Long Read Transposon Insertion-site Sequencing). This experiment data contains sequence files generated using Nanopore and Illumina platforms. Biotin1308.fastq.gz and Biotin2508.fastq.gz are fastq files generated from nanopore technology. Rep1-Tn.fastq.gz and Rep1-Tn.fastq.gz are fastq files generated using Illumina platform. In this study, we have compared the efficiency of two methods in identification of transposon insertion sites.
Project description:We identified exosomal miRNA biomarkers for pancreatic cancer diagnostics by isolating exosomes using a recently developed magnetic nanopore isolation technology and small RNA sequencing.
Project description:We optimized a protocol to enrich, digest and add poly(A) tail to the circular RNAs in order to make them compatible with the Oxfor Nanopore Technology for full-length sequencing
Project description:Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.
Project description:S. meliloti strains with a bi- and monopartite genome configuration were constructed by consecutive Cre/lox-mediated site-specific fusions of the secondary replicons. Beside the correct genomic arrangements, these strains and precursors were tested for variations in the nucleotide sequence. Futher, a marker fequency analysis was performed to test if replication is initiated at all origins and to determine the replication termination regions of the triple replicon fusion molecule. To gain the sequence data for these analyses, respective strains were applied to whole genome sequencing using an Illumina MiSeq-System and Oxford Nanopore (MinION) sequencing technology.
Project description:Replicon-seq is a method to study the progression of sister replisomes during DNA replication. This method relies excision of the full-length of replicons by the fusion of MNase to MCM4 and sequencing via Nanopore technology.
Project description:We sequenced DNA from a bulk of Col x Ler F2 hybrid plants (WT and recq4) using Nanopore long-read sequencing and identified crossover sites with COmapper. For nanopore sequencing of gDNA from 1,000 pooled seedlings, 10-day-old seedlings were ground in liquid nitrogen using a mortar and pestle. The ground tissue was resuspended in four volumes of CTAB buffer (1% [w/v] CTAB, 50 mM Tris-HCl pH 8.0, 0.7 M NaCl, 10 mM EDTA) and incubated at 65°C for 30 min. Following chloroform extraction, isopropanol precipitation and removal of RNAs as above, the gDNA pellet was resuspended in 150 μl TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) buffer and gDNA was quantified using a Qubit dsDNA Broad Range assay kit (Thermo Fisher, Q32853). Nine micrograms of gDNA from pollen or seedlings was used to construct a nanopore long-read sequencing library using a Ligation Sequencing Kit V14 (Nanopore, SQK-LSK114). The libraries were sequenced using a PromethION platform (BGI, Hong Kong).
Project description:We have used the genetic resources of Arabidopsis thaliana to generate mutant lines that have reactivated TE expression. We used these lines with long-read Oxford Nanopore sequencing technology to capture Transposable Element (TE) mRNAs for TE transcript annotation.
Project description:We explored changes at gene-level or transcript-level in embryonic stem cells, before and after in vitro differentiation with retinoic acid. RNA was sequenced both via Illumina short reads, and with Oxford Nanopore Technology with cDNA and direct RNA sequencing.
Project description:We explored changes at gene-level or transcript-level in embryonic stem cells, before and after in vitro differentiation with retinoic acid. RNA was sequenced both via Illumina short reads, and with Oxford Nanopore Technology with cDNA and direct RNA sequencing.