Biology subjects

Olsen, H. E.

Publications and source records attributed to Olsen, H. E..

4 recordsLinked to original sources

bioRxiv2017

Linear Assembly of a Human Y Centromere using Nanopore Long Reads

The human genome reference sequence remains incomplete due to the challenge of assembling long tracts of near-identical tandem repeats-in centromeric regions. To address this, we have implemented a nanopore sequencing strategy to generate high quality reads that span hundreds of kilobases of highly repetitive DNAs. Here, we use this advance to produce a sequence assembly and characterization of the centromeric region of a human Y chromosome.

genomics↗

bioRxiv2017

Nanopore sequencing and assembly of a human genome with ultra-long reads

Nanopore sequencing is a promising technique for genome sequencing due to its portability, ability to sequence long reads from single molecules, and to simultaneously assay DNA methylation. However until recently nanopore sequencing has been mainly applied to small genomes, due to the limited output attainable. We present nanopore sequencing and assembly of the GM12878 Utah/Ceph human reference genome generated using the Oxford Nanopore MinION and R9.4 version chemistry. We generated 91.2 Gb of sequence data ([~]30x theoretical coverage) from 39 flowcells. De novo assembly yielded a highly complete and contiguous assembly (NG50 [~]3Mb). We observed considerable variability in homopolymeric tract resolution between different basecallers. The data permitted sensitive detection of both large structural variants and epigenetic modifications. Further we developed a new approach exploiting the long-read capability of this system and found that adding an additional 5x-coverage of ultra-long reads (read N50 of 99.7kb) more than doubled the assembly contiguity. Modelling the repeat structure of the human genome predicts extraordinarily contiguous assemblies may be possible using nanopore reads alone. Portable de novo sequencing of human genomes may be important for rapid point-of-care diagnosis of rare genetic diseases and cancer, and monitoring of cancer progression. The complete dataset including raw signal is available as an Amazon Web Services Open Dataset at: https://github.com/nanopore-wgs-consortium/NA12878.

genomics↗

bioRxiv2017

Nanopore Long-Read RNAseq Reveals Widespread Transcriptional Variation Among the Surface Receptors of Individual B cells

Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single cell level. Short-read RNAseq, while the current standard for gene expression quantification, is limited in its ability to resolve complex isoforms because it fails to sequence full-length cDNA copies of RNA molecules. Here, we investigated whether RNAseq using the long-read single-molecule Oxford Nanopore MinION sequencing technology (ONT RNAseq) would be able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. After successfully benchmarking our experimental and computational approaches on a mixture of synthetic transcripts, we analyzed individual murine B1a cells using a new cellular indexing strategy. Using the Mandalorion analysis pipeline we developed, we identified thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. We also identified hundreds of genes expressed across B1a cells that displayed multiple complex isoforms, including several B cell specific surface receptors and the antibody heavy chain (IGH) locus. Our results show that not only can we identify complex isoforms, but also quantify their expression, at the single cell level.

genomics↗

bioRxiv2017

Whole genome sequencing and assembly of a Caenorhabditis elegans genome with complex genomic rearrangements using the MinION sequencing device

Advances in 3rd generation sequencing have opened new possibilities for benchtop whole genome sequencing. The MinION is a portable device that uses nanopore technology and can sequence long DNA molecules. MinION long reads are well suited for sequencing and de novo assembly of complex genomes with large repetitive elements. Long reads also facilitate the identification of complex genomic rearrangements such as those observed in tumor genomes. To assess the feasibility of the de novo assembly of large complex genomes using both MinION and Illumina platforms, we sequenced the genome of a Caenorhabditis elegans strain that contains a complex acetaldehyde-induced rearrangement and a biolistic bombardment-mediated insertion of a GFP containing plasmid. Using [~]5.8 gigabases of MinION sequence data, we were able to assemble a C. elegans genome containing 145 contigs (N50 contig length = 1.22 Mb) that covered >99% of the 100,286,401 bp reference genome. In contrast, using [~]8.04 gigabases of Illumina sequence data, we were able to assemble a C. elegans genome in 38,645 contigs (N50 contig length = [~]26 kb) containing 117 Mb. From the MinION genome assembly we identified the complex structures of both the acetaldehyde-induced mutation and the biolistic-mediated insertion. To date, this is the largest genome to be assembled exclusively from MinION data and is the first demonstration that the long reads of MinION sequencing can be used for whole genome assembly of large (100 Mb) genomes and the elucidation of complex genomic rearrangements.

genomics↗