Mon 11th November 2019

Hear about the latest tech updates for Oxford Nanopore as well as hearing from local users about their latest work using nanopore technology. 

Some talks will be given in Korean and some in English. Translation will not be provided.

Confirmed speakers include:

• Jangsup Moon, Seoul National University Hospital
• Hui-Su Kim, KOGIC, UNIST
• Sang-Choon Lee, PHYZEN
• Yong Min Kim, Korea Research Institute of Bioscience & Biotechnology (KRIBB)
• Jungwoo Lee, Cancer Genome Research Center
• Seong Wook Yang, Yonsei University

There is no delegate fee for this event. 
Your place at this event will be confirmed via email from events@nanoporetech.com. Completion of this form does not constitute confirmation. Spaces are limited and will be allocated on a first-come, first-served basis.

Rapid pathogen identification is very important in the treatment of infectious disease. Despite the current efforts to diagnose pathogens, in many cases, they remain undiagnosed. Nanopore sequencing contains many advantages for metagenomics analysis, which are simple library preparation procedure, real-time analysis, and long-read sequencing. We have performed nanopore sequencing in hundreds of clinical samples obtained from various types of infectious disease patients treated at Seoul National University Hospital. In this talk, I will demonstrate the usefulness and benefits of nanopore metagenomics sequencing for the identification of pathogens in the clinical setting. Particularly for bacterial detection, the culture-independent nanopore metagenomics sequencing was faster and more sensitive than conventional culture studies and was capable of polymicrobial detection at once. Nanopore metagenomic sequencing can be of great help to the clinical treatment of patients with infectious diseases through subsequent studies.

Long DNA reads produced by single molecule and pore-based sequencers are more suitable for assembly and structural variation discovery than short read DNA fragments. For de novo assembly, PacBio and Oxford Nanopore Technologies (ONT) are favorite options. However, PacBio’s SMRT sequencing is expensive for a full human genome assembly and costs over 40,000 USD for 30x coverage as of 2019. ONT PromethION sequencing, on the other hand, is one-twelfth the price of PacBio for the same coverage. This study aimed to compare the cost-effectiveness of ONT PromethION and PacBio’s SMRT sequencing in relation to the quality. We performed whole genome de novo assemblies and comparison to construct an improved version of KOREF, the Korean reference genome, using sequencing data produced by PromethION and PacBio. With PromethION, an assembly using sequenced reads with 64x coverage (193 Gb, 3 flowcell sequencing) resulted in 3,725 contigs with N50s of 16.7 Mbp and a total genome length of 2.8 Gbp. It was comparable to a KOREF assembly constructed using PacBio at 62x coverage (188 Gbp, 2,695 contigs and N50s of 17.9 Mbp). When we applied Hi-C-derived long-range mapping data, an even higher quality assembly for the 64x coverage was achieved, resulting in 3,179 scaffolds with an N50 of 56.4 Mbp. The pore-based PromethION approach provides a good quality chromosome-scale human genome assembly at a low cost with long maximum contig and scaffold lengths, and is more cost-effective than PacBio at comparable quality measurements.

Nanopore sequencing technology of Oxford Nanopore Technologies (ONT) is the latest third generation sequencing technology for cost-effective high-throughput sequencing of long DNA molecules. At present, this technology is providing new opportunity for efficient and high-quality genome analysis and well demonstrated by more than 300 genomes of various organisms assembled using Nanopore sequencing data. So far, our company sequenced genomes of more than 170 samples and produced total >900 Gb Nanopore sequencing data in Korea. Using the data, we de novo assembled and generated high-quality draft genome sequences of bacteria, fungi, mushrooms, insects and plants. In this talk, we will introduce sequencing and genome assembly results obtained by Nanopore sequencing and bioinformatics analysis of our company.  

Construction of high-quality genome in plants is an essential and fundamental analysis for further application of genome information from basic science to breeding. However, the high content of repetitive sequences and polyploidy are barriers to constructing a high-quality reference genome. In the case of polyploid species containing large amount of duplicated regions, short reads can produce chimeric sequences and fragmented contigs. Long or ultra-long reads from third-generation sequencing platforms can be a solution for constructing high-quality reference genomes for polyploid species. ONT technology is one of the candidates to quickly and cost-effectively generate longer read length that can facilitate the assembly of complex plant genomes. The read length depends on the physical length of the DNA, which directly affects the sequencing quality. However, extraction of high purity and high molecular DNA have been difficult in plants because of high content of polysaccharides and polyphenols. Therefore, the establishment of DNA extraction method is the most critical step to leverage the value of nanopore sequencing platforms. Here, we report improved sequencing results and genome assemblies of two plant genomes, Solanum lycopersicum Hawaii7996 and Hibiscus syriacus Gangneung.  

With the advent of next-generation sequencing (NGS) technology, researchers have been able to capture sequences from the genome for their interest of study. Nonetheless, universal sequencing technology such as Illumina, still contains the insufficient contextual information due to their usage of short fragmented reads, which limits understanding of the complex structures of the genome and transcriptome. Oxford Nanopore Technology (ONT) on the other hand, has allowed the researchers to capture the full-length transcripts, thereby ensuring the full and novel information of transcriptome. Although a large number of short-read based analyses have accumulated the cancer transcriptome profile, the attempts of long-read based profiling for a better understanding of cancer are still in need. To meet this need, we have sequenced 20 pairs of Korean colon cancer patients using cDNA-PCR Nanopore sequencing. We identified the long-read based complex transcriptome profiles and selected colon cancer specific isoforms, as well as fusion gene candidates. We also selected differentially expressed transcript candidates including non-coding RNAs to reveal their molecular and functional analogy which could explain the clinical implication in colon cancer. Overall, we have characterized the transcriptome profiles of Korean colon cancer patients using Nanopore sequencing to identify a potential biomarker that can be used in the clinical field. Using such technology with further validation, we expect to find novel and more precise clinical biomarkers in colon cancer which could lead to a new molecular mechanism to better understand colon carcinogenesis.  

Next-generation sequencing (NGS) technologies have accelerated the study of small RNAs (sRNAs) on a genome-wide scale. However, existing sRNA library preparation methods for sequencing technology entails serious bias and non-specific primer dimerization, mainly during adapter ligation steps and adaptor-based PCR amplification steps. The successive rate of adaptor ligation steps is sorely dependent on the experimental proficiency, and the maximum rate is less than 70% of total sRNAs. Furthermore, several types of sRNA, including plant microRNAs (miRNA), small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNA) in mammals, and small interfering RNAs (siRNA) contain a 2’-O-methyl (2’-OMe) modification at their 3′ terminal nucleotide. This particularity hinders 3′ adapter ligation and makes the construction of small RNA libraries highly challenging. To overcome these problems, we here suggest the XENO sensor method that uses XENO nucleotides to recognize small RNAs without going through adaptor ligations steps. The XENO sensors are highly specific enough to restore 95% of given sRNAs and generate no primer dimer background during amplification steps. This novel method is highly efficient in profiling small RNAs without bias and could be suitable for the Nanopore platform to sequence small RNAs.