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Nanopore Day, San Diego

Wed 4th March 2020

San Diego, CA, United States

Hear about the latest tech updates from Oxford Nanopore Technologies, as well as talks from scientists using nanopore sequencing in their work. 

There is no delegate fee for this event. 

Your place at this event will be confirmed via email from events@nanoporetech.com. Spaces are limited and will be allocated on a first-come, first-served basis.

Confirmed speakers include:

  • Todd Michael, JCVI
  • Brad Abramson, JCVI
  • Daniel Lorenz, UC San Diego
  • Karen Miga, UC Santa Cruz
  • Roger Volden, UC Santa Cruz

Human genome sequencing on the PromethION - Todd Michael, JCVI

The publication of the first diploid human genome established the need for de novo assembled genomes for accurate identification and annotation of human variation (Levy et al., 2007). Similar efforts revealed the same conclusions albeit with lower quality de novo assemblies (Wang et al., 2008; Wheeler et al., 2008). However, the cost and effort to de novo sequence the human genome has restricted broad adoption of this strategy and instead most efforts have focused on resequencing whole genomes or exomes using short (~100 bp) sequencing technologies (1000 Genomes Project Consortium, 2011, 2012, 2015; Telenti et al., 2016). It is estimated 19-40 Mb of sequence may be missing in the NCBI human reference genome due to ethnic and private differences within the human population, as well as the lack of diploid representation, or phasing of haplotypes (Li et al., 2010). For instance, in our HuRef diploid genome we found that non-SNP DNA variation only accounted for 22% of differences with the NCBI reference but made up 74% of all variant bases. In addition, when we were able to phase over 50% of HuRef into long haplotype blocks (>200 kb) we found that 44% of genes were heterozygous for one or more variants (Levy et al., 2007). Here we leverage the Oxford Nanopore Technologies (ONT) PromethION to rapidly generate an updated HuRef assembly.

 

Duckweed Gene Cycling Behavior Over Diel Cycles Using Nanopore Full Length cDNA Sequencing - Brad Abramson, JCVI

While long read nanopore sequencing has ushered in a new era of contiguous genome assemblies, accurate and complete gene predictions remain a challenge especially for unique species in underrepresented regions of the tree of life. Nanopore sequencing enables full length cDNA (FL-cDNA) sequencing that provides essential empirical evidence of intron/exon structures and alternative splicing, which compliments ab initio efforts to define gene models. Here we coupled time-of-day (TOD) sampling with FL-cDNA sequencing to improve the sensitivity and precision of gene calls in Duckweed. The Greater Duckweed, Spirodela polyrhiza is re-emerging as a model plant due to its extremely fast growth rate, aquatic lifestyle, small genome, minimal set of genes, transformation system, and reduced body plan. These results highlight the utility of using FL-cDNA to accurately annotate genomes and determine gene expression patterns.

 

Direct RNA sequencing enables m6A detection - Daniel Lorenz, UCSD

Direct RNA sequencing holds great promise for the de novo identification of RNA modifications at single-coordinate resolution; however, interpretation of raw sequencing output to discover modified bases remains a challenge. Using Oxford Nanopore’s direct RNA sequencing technology, we developed a random forest classifier trained using experimentally detected N6-methyladenosine (m6A) sites within DRACH motifs. Our software MINES (m6A Identification using Nanopore Sequencing) enables m6A annotation at single coordinate–level resolution from direct RNA nanopore sequencing.

 

The new era of telomere-to-telomere genomics: expanding studies of hidden genetic variation and function in the uncharted regions of the human genome - Karen Miga, UCSC

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts initially on the human X chromosome, we reconstructed the ∼3.1 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.

 

Highly Multiplexed Single-Cell Full-Length cDNA Sequencing of human immune cells with 10X Genomics and R2C2 - Roger Volden, UCSC

Single cell transcriptome analysis elucidates facets of cell biology that have been previously out of reach. However, the high-throughput analysis of thousands of single cell transcriptomes has been limited by sample preparation and sequencing technology. High-throughput single cell analysis today is facilitated by protocols like the 10X Genomics platform or Drop-Seq which generate cDNA pools in which the origin of a transcript is encoded at its 5’ or 3’ end. These cDNA pools are currently analyzed by short read Illumina sequencing which can identify the cellular origin of a transcript and what gene it was transcribed from. However, these methods fail to retrieve isoform information. In principle, cDNA pools prepared using these approaches can be analyzed with Pacific Biosciences and Oxford Nanopore long-read sequencers to retrieve isoform information but all current implementations rely heavily on Illumina short-reads for the analysis in addition to long reads. Here, we used R2C2 to sequence and demultiplex 9 million full-length cDNA molecules generated by the 10X Chromium platform from ∼3000 peripheral blood mononuclear cells (PBMCs). We used these reads to – independent from Illumina data – cluster cells into B cells, T cells, and Monocytes and generate isoform-level transcriptomes for these cell-types. We also generated isoform-level transcriptomes for all single cells and used this information to identify a wide range of isoform diversity between genes. Finally, we also designed a computational workflow to extract paired adaptive immune receptor – T cell receptor and B cell receptor (TCR and BCR) –sequences unique to each T and B cell. This work represents a new, simple, and powerful approach that –using a single sequencing method – can extract an unprecedented amount of information from thousands of single cells.

Event category:
Nanopore events

Type of Event:
Nanopore Day

Admission:
Registration