Hyojun Han

‘Shotgun DNA synthesis’ for the high-throughput construction of large DNA molecules

We developed a highly scalable ‘shotgun’ DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20u2009bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.

A fluorescence selection method for accurate large-gene synthesis.

The fundamental problem for low‐cost gene synthesis is errors that occur during the synthetic process. To address this problem, we developed a practical method that exploits the fact that the predominant errors are deletions. In this method, a simple fluorescence‐based readout was used to distinguish error‐free synthetic DNA molecules. To do this, we constructed vectors that contained multiple cloning sites and GFP. In the vectors, the GFP gene is designed to be out‐of‐frame, but insertion of an in‐framed synthetic DNA construct into the appropriate cloning site will lead to fluorescent cell colonies. We successfully used this method to synthesize five genes and improved the bp per error from 629 to 6552 by selecting green fluorescent colonies.

Targeted next-generation sequencing for comprehensive genetic profiling of pharmacogenes

Phenotypic differences in drug responses have been associated with known pharmacogenomic loci, but many remain to be characterized. Therefore, we developed next‐generation sequencing (NGS) panels to enable broad and unbiased inspection of genes that are involved in pharmacokinetics (PKs) and pharmacodynamics (PDs). These panels feature repetitively optimized probes to capture up to 114 PK/PD‐related genes with high coverage (99.6%) and accuracy (99.9%). Sequencing of a Korean cohort (n = 376) with the panels enabled profiling of actionable variants as well as rare variants of unknown functional consequences. Notably, variants that occurred at low frequency were enriched with likely protein‐damaging variants and previously unreported variants. Furthermore, in vitro evaluation of four pharmacogenes, including cytochrome P450 2C19 (CYP2C19), confirmed that many of these rare variants have considerable functional impact. The present study suggests that targeted NGS panels are readily applicable platforms to facilitate comprehensive profiling of pharmacogenes, including common but also rare variants that warrant screening for personalized medicine.

Association between mutations of critical pathway genes and survival outcomes according to the tumor location in colorectal cancer

Colorectal cancer (CRC) develops through the alteration of several critical pathways. This study was aimed at evaluating the influence of critical pathways on survival outcomes for patients with CRC.

Model for uniaxial compaction of ceramic powders

Abstract A phenomenological yield criterion has been proposed for modelling the uniaxial compaction processes of various ceramic powder compacts on the basis of continuum mechanics. It includes three parameters to characterise the geometric hardening of the powder compact and the strength of the base material. The model was applied to uniaxial compaction of three ceramic powders which possess different particle size distribution, particle morphology, and base material property. The values of parameters in the yield criterion were determined through the uniaxial compaction experiment. Using the yield criterion, the elastoplastic finite element calculation was carried out to analyse the compaction of the three ceramic powders.

Finite element simulation of duplex compaction processes

Abstract Three types of duplex compaction process have been simulated by the finite element method using a backward Euler method and a yield criterion, which can describe the densification behaviour of ceramic powder. The effects of two parameters in the yield criterion on the uniaxial compaction curve are examined. The friction force during a combination of inner and outer compaction can be significantly reduced by an intentional clearance between the inner and outer compacts. The calculated shape of duplex compacts and density distribution show good agreement with experimental results using ZrO2 powder. A height difference between inner and outer compacts after combination disappears by friction force reduction.

Multiple target loci assembly sequencing (mTAS)

Here we present multiple target loci assembly sequencing (mTAS), a method for examining multiple genomic loci in a single DNA sequencing read. The key to the success of mTAS target sequencing is the uniform amplification of multiple target genomic loci into a single DNA fragment using polymerase cycling assembly (PCA). Using this strategy, we successfully collected multiloci sequence information from a single DNA sequencing run. We applied mTAS to examine 29 different sets of human genomic loci, each containing from 2 to 11 single-nucleotide polymorphisms (SNP) present at different exons. We believe mTAS can be used to reduce the cost of Sanger sequencing-based genetic analysis.

Highly selective retrieval of accurate DNA utilizing a pool of in situ-replicated DNA from multiple next-generation sequencing platforms

Abstract Scalable and cost-effective production of error-free DNA is critical to meet the increased demand for such DNA in the field of biological science. Methods based on ‘Dial-out PCR’ have enabled the high-throughput error-free DNA synthesis from a microarray-synthesized DNA pool by labeling with retrieval PCR tags, and retrieving error-free DNA of which the sequence is identified via next generation sequencing (NGS). However, most of the retrieved products contain byproducts due to background amplification of redundantly labeled DNAs. Here, we present a highly selective retrieval method of desired DNA from a pool of millions of DNA clones from NGS platforms. Our strategy is based on replicating entire sequence-verified DNA molecules from NGS plates to obtain population-controlled DNA pool. Using the NGS-replica pool, we could perform improved and selective retrieval of desired DNA from the replicated DNA pool compared to other dial-out PCR based methods. To evaluate the method, we tested this strategy by using 454, Illumina, and Ion Torrent platforms for producing NGS-replica pool. As a result, we observed a highly selective retrieval yield of over 95%. We anticipate that applications based on this method will enable the preparation of high-fidelity sequenced DNA from heterogeneous collections of DNA molecules.

Abstract A72: Mutation signature associated with colorectal cancer prognosis identified by targeted next-generation sequencing

Background Although genomic data of CRC continue to accumulate, only limited information on prognostic role of the alterations have been reported. We have analyzed the prognostic impact of the key mutations in CRC using next generation sequencing technology. Methods We selected 40 genes from 5 critical pathways (WNT, TGF-B, PI3K, RTK-RAS and P53) of CRC based on TCGA data. Homogenous population of CRC patients were used to investigate the prognostic implication: 188 stage III or high-risk stage II CRC patients treated with curative surgery followed by adjuvant 5-fluorouracil/ leucovorin/oxaliplatin (FOLFOX) chemotherapy. Archival tissue from Seoul National University Hospital Tumor Bank was used. The target (size 109kb) enrichment process was proceeded base on in-solution hybridization with biotinylated probes. The captured library was amplified and sequenced using Hiseq 2500 (Illumina, USA). Sequencing data was aligned to GRCh37 and variant call and somatic analysis processes was performed by VarScan2 and were annotated with ANNOVAR. Using somatic nonsynonymous mutation data, we selected mutant gene signature which is associated with prognosis. We used binary collapsing method in a forward stepwise selection method to combine mutated genes. Test statistics were obtained from Cox-proportional hazard model. Two-sided p-values of less than 0.05 were considered statistically significant. Results Among a total of 188 patients, 63 had tumor in proximal location and 125 had tumor in distal location. Tumor stage was high-risk stage II in 21 patients and III in 167 patients. Average coverage of the total samples was 417X (414X tumor and 420X normal mucosa). Mutation frequencies were similar with the TCGA data except for NRAS and DKK2, having lower frequency in the present study. During a median follow-up duration of 58 months, 45 relapse and 23 death events have occurred. Alteration in individual gene or pathway did not have prognostic significance in terms of disease free survival (DFS) and overall survival (OS). By using binary collapsing method, we identified a 4 gene signature comprising ACVR1B, ERBB2, LRP5, and KRAS. 89 patients (47.3%) had 1 or more mutation in these 4 genes. 4 gene mutant group had worse DFS and OS compared with 4 gene wild type group. 3-year DFS and 5-year OS was 72.8% and 82.5% in mutant group compared with 88.7% and 96.0% in wild type group (p-values 0.004 and 0.001, respectively). Multivariate analysis revealed 4 gene signature as an independent negative prognostic factor of DFS and OS (adjusted hazard ratios 2.48 and 4.26, respectively). Conclusion Mutations in the 4 genes (ACVR1B, ERBB2, LRP5, and KRAS) were associated with poor prognosis in CRC patients treated with surgery followed by adjuvant FOLFOX chemotherapy. Validation study in an independent cohort is currently underway. Citation Format: Dae-Won Lee, Yongjun Cha, Sae-Won Han, Si-Hyun Lee, Hwang-Phill Kim, Jaemyun Lyu, Hyojun Han, Hyoki Kim, Hoon Jang, Duhee Bang, Iksoo Huh, Taesung Park, Jeong Mo Bae, Jae-Kyung Won, Seung-Yong Jeong, Kyu Joo Park, Gyeong Hoon Kang, Tae-You Kim. Mutation signature associated with colorectal cancer prognosis identified by targeted next-generation sequencing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A72.

Analytic Model for Non-Steady State Heat Transfer of Powder Pressing Roller

An analysis for non steady state heat transfer of a hot pressing roller was suggested in 1-dimensional model. The surface temperature on hot pressing roller was predicted by using surface contact heat transfer coefficient calculated with induced analytic solution. We calculated the size of iron powder, influencing on surface contact heat transfer coefficient. Since coarse iron powder has reduced heat transfer coefficient during contacting on roll surface with smaller contact area, temperature on roller surface has been expected to decrease. This predicted temperature by the analytic model was fairly reasonable in comparison with experimental data and finite element model.