Project description:Oxford Nanopore (ONT) is a leading long-read technology which has been revolutionizing transcriptome analysis through its capacity to sequence the majority of transcripts from end-to-end. This has greatly increased our ability to study the diversity of transcription mechanisms such as transcription initiation, termination, and alternative splicing. However, ONT still suffers from high error rates which have thus far limited its scope to reference-based analyses. When a reference is not available or is not a viable option due to reference-bias, error correction is a crucial step towards the reconstruction of the sequenced transcripts and downstream sequence analysis of transcripts. In this paper, we present a novel computational method to error correct ONT cDNA sequencing data, called isONcorrect. IsONcorrect is able to jointly use all isoforms from a gene during error correction, thereby allowing it to correct reads at low sequencing depths. We are able to obtain a median accuracy of 98.9-99.6%, demonstrating the feasibility of applying cost-effective cDNA full transcript length sequencing for reference-free transcriptome analysis.
Project description:Droplet-based single-cell sequencing techniques have provided unprecedented insight into cellular heterogeneities within tissues. However, these approaches only allow for the measurement of the distal parts of a transcript following short-read sequencing. Therefore, splicing and sequence diversity information is lost for the majority of the transcript. The application of long-read Nanopore sequencing to droplet-based methods is challenging because of the low base-calling accuracy currently associated with Nanopore sequencing. Although several approaches that use additional short-read sequencing to error-correct the barcode and UMI sequences have been developed, these techniques are limited by the requirement to sequence a library using both short- and long-read sequencing. Here we introduce a novel approach termed single-cell Barcode UMI Correction sequencing (scBUC-seq) to efficiently error-correct barcode and UMI oligonucleotide sequences synthesized by using blocks of dimeric nucleotides. The method can be applied to correct both short-read and long-read sequencing, thereby allowing users to recover more reads per cell that permits direct single-cell Nanopore sequencing for the first time. We illustrate our method by using species-mixing experiments to evaluate barcode assignment accuracy and multiple myeloma cell lines to evaluate differential isoform usage and Ewing’s sarcoma cells to demonstrate Ig fusion transcript analysis.
Project description:Higher-order chromatin structure arises from the combinatorial physical interactions of many genomic loci. To investigate this aspect of genome architecture we developed Pore-C, which couples chromatin conformation capture with Oxford Nanopore Technologies (ONT) long reads to directly sequence multi-way chromatin contacts without amplification.
Project description:The whole genome sequencing (WGS) has become a crucial tool in understanding genome structure and genetic variation. The MinION sequencing of Oxford Nanopore Technologies (ONT) is an excellent approach for performing WGS and it has advantages in comparison with other Next-Generation Sequencing (NGS): It is relatively inexpensive, portable, has simple library preparation, can be monitored in real-time, and has no theoretical limits on reading length. Sorghum bicolor (L.) Moench is diploid (2n = 2x = 20) with a genome size of about 730 Mb, and its genome sequence information is released in the Phytozome database. Therefore, sorghum can be used as a good reference. However, plant species have complex and large genomes when compared to animals or microorganisms. As a result, complete genome sequencing is difficult for plant species. MinION sequencing that produces long-reads can be an excellent tool for overcoming the weak assembly of short-reads generated from NGS by minimizing the generation of gaps or covering the repetitive sequence that appears on the plant genome. Here, we conducted the genome sequencing for S. bicolor cv. BTx623 while using the MinION platform and obtained 895,678 reads and 17.9 gigabytes (Gb) (ca. 25× coverage of reference) from long-read sequence data. A total of 6124 contigs (covering 45.9%) were generated from Canu, and a total of 2661 contigs (covering 50%) were generated from Minimap and Miniasm with a Racon through a de novo assembly using two different tools and mapped assembled contigs against the sorghum reference genome. Our results provide an optimal series of long-read sequencing analysis for plant species while using the MinION platform and a clue to determine the total sequencing scale for optimal coverage that is based on various genome sizes.
Project description:Neurons in posterior parietal cortex contribute to the execution of goal-directed navigation and other decision-making tasks. Although molecular studies have catalogued over fifty cortical cell types, it remains unknown what distinct functions they serve during goal-directed navigation. Here, we identified a molecularly defined subset of somatostatin (Sst) inhibitory neurons that, in mouse posterior parietal cortex, carry a novel cell type-specific error correction signal for navigation. We obtained repeatable experimental access to these cells using an adeno-associated virus (AAV) in which gene expression is driven by an enhancer that functions specifically in a subset of Sst cells. We found that during goal-directed navigation in a virtual environment, this subset of Sst neurons activates in a synchronous pattern that is distinct from the activity of surrounding neurons, including other Sst neurons. Using in vivo two-photon photostimulation and ex vivo paired patch clamp recordings, we show that nearby cells of this Sst subtype excite each other through gap junctions, revealing a self-excitation circuit motif that contributes to the synchronous activity of this cell type. Remarkably, these cells selectively activate as mice execute course corrections for deviations in their virtual heading during navigation toward a reward location, both for self- and experimentally-induced deviations. We propose that this subtype of Sst neurons provides a self-reinforcing and cell type-specific error-correction signal in posterior parietal cortex that may aid the execution and learning of accurate goal-directed navigation trajectories.
Project description:Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades. Recently, a nanopore-based sequencing instrument, the Oxford Nanopore MinION, has become available, and we used this for sequencing the Saccharomyces cerevisiae genome. To make use of these data, we developed a novel open-source hybrid error correction algorithm Nanocorr specifically for Oxford Nanopore reads, because existing packages were incapable of assembling the long read lengths (5-50 kbp) at such high error rates (between ∼5% and 40% error). With this new method, we were able to perform a hybrid error correction of the nanopore reads using complementary MiSeq data and produce a de novo assembly that is highly contiguous and accurate: The contig N50 length is more than ten times greater than an Illumina-only assembly (678 kb versus 59.9 kbp) and has >99.88% consensus identity when compared to the reference. Furthermore, the assembly with the long nanopore reads presents a much more complete representation of the features of the genome and correctly assembles gene cassettes, rRNAs, transposable elements, and other genomic features that were almost entirely absent in the Illumina-only assembly.