Highly Accurate Sequence- and Position-Independent Error Profiling of DNA Synthesis and Sequencing.

A comprehensive error analysis of DNA-stored data during processing, such as DNA synthesis and sequencing, is crucial for reliable DNA data storage. Both synthesis and sequencing errors depend on the sequence and the transition of bases of nucleotides; ignoring either one of the error sources leads to technical challenges in minimizing the error rate. Here, we present a methodology and toolkit that utilizes an oligonucleotide library generated from a 10-base-shifted sequence array, which is individually labeled with unique molecular identifiers, to delineate and profile DNA synthesis and sequencing errors simultaneously. This methodology enables position- and sequence-independent error profiling of both DNA synthesis and sequencing. Using this toolkit, we report base transitional errors in both synthesis and sequencing in general DNA data storage as well as degenerate-base-augmented DNA data storage. The methodology and data presented will contribute to the development of DNA sequence designs with minimal error.

Canine Somatic Mutations from Whole-Exome Sequencing of B-Cell Lymphomas in Six Canine Breeds-A Preliminary Study.

Canine lymphoma (CL) is one of the most common malignant tumors in dogs. The cause of CL remains unclear. Genetic mutations that have been suggested as possible causes of CL are not fully understood. Whole-exome sequencing (WES) is a time- and cost-effective method for detecting genetic variants targeting only the protein-coding regions (exons) that are part of the entire genome region. A total of eight patients with B-cell lymphomas were recruited, and WES analysis was performed on whole blood and lymph node aspirate samples from each patient. A total of 17 somatic variants (GOLIM4, ITM2B, STN1, UNC79, PLEKHG4, BRF1, ENSCAFG00845007156, SEMA6B, DSC1, TNFAIP1, MYLK3, WAPL, ADORA2B, LOXHD1, GP6, AZIN1, and NCSTN) with moderate to high impact were identified by WES analysis. Through a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 17 genes with somatic mutations, a total of 16 pathways were identified. Overall, the somatic mutations identified in this study suggest novel candidate mutations for CL, and further studies are needed to confirm the role of these mutations.

Pen-drawn Marangoni swimmer.

Pen-drawing is an intuitive, convenient, and creative fabrication method for delivering emergent and adaptive design to real devices. To demonstrate the application of pen-drawing to robot construction, we developed pen-drawn Marangoni swimmers that perform complex programmed tasks using a simple and accessible manufacturing process. By simply drawing on substrates using ink-based Marangoni fuel, the swimmers demonstrate advanced robotic motions such as polygon and star-shaped trajectories, and navigate through maze. The versatility of pen-drawing allows the integration of the swimmers with time-varying substrates, enabling multi-step motion tasks such as cargo delivery and return to the original place. We believe that our pen-based approach will significantly expand the potential applications of miniaturized swimming robots and provide new opportunities for simple robotic implementations.

I-LIFT (image-based laser-induced forward transfer) platform for manipulating encoded microparticles.

Encoded microparticles have great potential in small-volume multiplexed assays. It is important to link the micro-level assays to the macro-level by indexing and manipulating the microparticles to enhance their versatility. There are technologies to actively manipulate the encoded microparticles, but none is capable of directly manipulating the encoded microparticles with homogeneous physical properties. Here, we report the image-based laser-induced forward transfer system for active manipulation of the graphically encoded microparticles. By demonstrating the direct retrieval of the microparticles of interest, we show that this system has the potential to expand the usage of encoded microparticles.

Association of B cell profile and receptor repertoire with the progression of Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent type of dementia. Reports have revealed that the peripheral immune system is linked to neuropathology; however, little is known about the contribution of B lymphocytes in AD. For this longitudinal study, 133 participants are included at baseline and second-year follow-up. Also, we analyze B cell receptor (BCR) repertoire data generated from a public dataset of three normal and 10 AD samples and perform BCR repertoire profiling and pairwise sharing analysis. As a result, longitudinal increase in B lymphocytes is associated with increased cerebral amyloid deposition and hyperactivates induced pluripotent stem cell-derived microglia with loss-of-function for beta-amyloid clearance. Patients with AD share similar class-switched BCR sequences with identical isotypes, despite the high somatic hypermutation rate. Thus, BCR repertoire profiling can lead to the development of individualized immune-based therapeutics and treatment. We provide evidence of both quantitative and qualitative changes in B lymphocytes during AD pathogenesis.

Purification of multiplex oligonucleotide libraries by synthesis and selection.

Complex oligonucleotide (oligo) libraries are essential materials for diverse applications in synthetic biology, pharmaceutical production, nanotechnology and DNA-based data storage. However, the error rates in synthesizing complex oligo libraries can be substantial, leading to increment in cost and labor for the applications. As most synthesis errors arise from faulty insertions and deletions, we developed a length-based method with single-base resolution for purification of complex libraries containing oligos of identical or different lengths. Our method-purification of multiplex oligonucleotide libraries by synthesis and selection-can be performed either step-by-step manually or using a next-generation sequencer. When applied to a digital data-encoded library containing oligos of identical length, the method increased the purity of full-length oligos from 83% to 97%. We also show that libraries encoding the complementarity-determining region H3 with three different lengths (with an empirically achieved diversity >106) can be simultaneously purified in one pot, increasing the in-frame oligo fraction from 49.6% to 83.5%.

Advances in Tumor Sampling and Sequencing in Breast Cancer and their Application in Precision Diagnostics and Therapeutics.

Intra- and Inter-tumoral heterogeneity is one of the main hurdles in diagnosing and treating breast cancer. Selecting, sampling, and sequencing the samples appropriately provide unique opportunities in realizing precision medicine. This chapter reviews some of the past landmarks, state-of-the-art technologies, and future directions of translational research in terms of tumor sampling technologies and sequencing in breast cancer. In the state-of-the-art technologies section, the technologies are categorized in terms of scientific, precision diagnostic, and precision therapeutic tools. Finally, limitations and future directions regarding various translational research for clinical applications using these technologies will be discussed.

Direct 2D-to-3D transformation of pen drawings.

Pen drawing is a method that allows simple, inexpensive, and intuitive two-dimensional (2D) fabrication. To integrate such advantages of pen drawing in fabricating 3D objects, we developed a 3D fabrication technology that can directly transform pen-drawn 2D precursors into 3D geometries. 2D-to-3D transformation of pen drawings is facilitated by surface tension-driven capillary peeling and floating of dried ink film when the drawing is dipped into an aqueous monomer solution. Selective control of the floating and anchoring parts of a 2D precursor allowed the 2D drawing to transform into the designed 3D structure. The transformed 3D geometry can then be fixed by structural reinforcement using surface-initiated polymerization. By transforming simple pen-drawn 2D structures into complex 3D structures, our approach enables freestyle rapid prototyping via pen drawing, as well as mass production of 3D objects via roll-to-roll processing.

Cell-Free Bacteriophage Genome Synthesis Using Low-Cost Sequence-Verified Array-Synthesized Oligonucleotides.

Synthesizing engineered bacteriophages (phages) for human use has potential in various applications ranging from drug screening using a phage display to clinical use using phage therapy. However, the engineering of phages conventionally involves the use of an in vivo system that has low production efficiency because of high virulence against the host and low transformation efficiency. To circumvent these issues, de novo phage genome synthesis using chemically synthesized oligonucleotides (oligos) has increased the potential for engineering phages in a cell-free system. Here, we present a cell-free, low-cost, de novo gene synthesis technology called Sniper assembly for phage genome construction. With massively parallel sequencing of microarray-synthesized oligos, we generated and identified approximately 100 000 clonal DNA clusters in vitro and 5000 error-free ones in a cell-free environment. To demonstrate its practical application, we synthesized the Acinetobacter phage AP205 genome (4268 bp) using 65 sequence-verified DNA clones. Compared to previous reports, Sniper assembly lowered the genome synthesis cost ($0.0137/bp) by producing low-cost sequence-verified DNA.

Microspinning: Local Surface Mixing via Rotation of Magnetic Microparticles for Efficient Small-Volume Bioassays.

The need for high-throughput screening has led to the miniaturization of the reaction volume of the chamber in bioassays. As the reactor gets smaller, surface tension dominates the gravitational or inertial force, and mixing efficiency decreases in small-scale reactions. Because passive mixing by simple diffusion in tens of microliter-scale volumes takes a long time, active mixing is needed. Here, we report an efficient micromixing method using magnetically rotating microparticles with patterned magnetization induced by magnetic nanoparticle chains. Because the microparticles have magnetization patterning due to fabrication with magnetic nanoparticle chains, the microparticles can rotate along the external rotating magnetic field, causing micromixing. We validated the reaction efficiency by comparing this micromixing method with other mixing methods such as simple diffusion and the use of a rocking shaker at various working volumes. This method has the potential to be widely utilized in suspension assay technology as an efficient mixing strategy.

Barcode-free next-generation sequencing error validation for ultra-rare variant detection.

The advent of next-generation sequencing (NGS) has accelerated biomedical research by enabling the high-throughput analysis of DNA sequences at a very low cost. However, NGS has limitations in detecting rare-frequency variants (< 1%) because of high sequencing errors (> 0.1~1%). NGS errors could be filtered out using molecular barcodes, by comparing read replicates among those with the same barcodes. Accordingly, these barcoding methods require redundant reads of non-target sequences, resulting in high sequencing cost. Here, we present a cost-effective NGS error validation method in a barcode-free manner. By physically extracting and individually amplifying the DNA clones of erroneous reads, we distinguish true variants of frequency > 0.003% from the systematic NGS error and selectively validate NGS error after NGS. We achieve a PCR-induced error rate of 2.5×10-6 per base per doubling event, using 10 times less sequencing reads compared to those from previous studies.

One-Step Generation of a Drug-Releasing Hydrogel Microarray-On-A-Chip for Large-Scale Sequential Drug Combination Screening.

Large-scale screening of sequential drug combinations, wherein the dynamic rewiring of intracellular pathways leads to promising therapeutic effects and improvements in quality of life, is essential for personalized medicine to ensure realistic cost and time requirements and less sample consumption. However, the large-scale screening requires expensive and complicated liquid handling systems for automation and therefore lowers the accessibility to clinicians or biologists, limiting the full potential of sequential drug combinations in clinical applications and academic investigations. Here, a miniaturized platform for high-throughput combinatorial drug screening that is "pipetting-free" and scalable for the screening of sequential drug combinations is presented. The platform uses parallel and bottom-up formation of a heterogeneous drug-releasing hydrogel microarray by self-assembly of drug-laden hydrogel microparticles. This approach eliminates the need for liquid handling systems and time-consuming operation in high-throughput large-scale screening. In addition, the serial replacement of the drug-releasing microarray-on-a-chip facilitates different drug exchange in each and every microwell in a simple and highly parallel manner, supporting scalable implementation of multistep combinatorial screening. The proposed strategy can be applied to various forms of combinatorial drug screening with limited amounts of samples and resources, which will broaden the use of the large-scale screening for precision medicine.

Monozygotic twins with shared de novo GATA2 mutation but dissimilar phenotypes due to differential promoter methylation.

A revised WHO classification of hematopoietic neoplasm introduced the new category 'Myeloid Neoplasms with Germline Predisposition', reflecting the growing importance of genetic testing for myeloid neoplasms. Here, we investigated monozygotic twins with the same de novo mutation in GATA2 but different phenotypes. The patient suffering a bleeding tendency was diagnosed with myelodysplastic syndrome (MDS), and her monozygotic twin showed dysmegakaryopoietic features in the bone marrow. Targeted sequencing revealed the same germline mutation in GATA2, c.1192C > T, in both sisters and different somatic mutations in 14 genes between the sisters. The GATA2 mutation was absent in both parents, and their hemograms were normal. The methylation profile of the GATA2 promoter region was different between the twins, showing denser promoter methylation in the patient, correlated with MDS. Thus, we concluded that the twins had acquired a de novo GATA2 mutation but showed different phenotypes, possibly due to the critical role of epigenetic changes.

PHLI-seq: constructing and visualizing cancer genomic maps in 3D by phenotype-based high-throughput laser-aided isolation and sequencing.

Spatial mapping of genomic data to tissue context in a high-throughput and high-resolution manner has been challenging due to technical limitations. Here, we describe PHLI-seq, a novel approach that enables high-throughput isolation and genome-wide sequence analysis of single cells or small numbers of cells to construct genomic maps within cancer tissue in relation to the images or phenotypes of the cells. By applying PHLI-seq, we reveal the heterogeneity of breast cancer tissues at a high resolution and map the genomic landscape of the cells to their corresponding spatial locations and phenotypes in the 3D tumor mass.

Hierarchical shape-by-shape assembly of microparticles for micrometer-scale viral delivery of two different genes.

Understanding tissue engineering using a bottom-up approach has been hindered by technical limitations because no platform can demonstrate the controlled formation of a heterogeneous population of cells in microscale. Here, we demonstrate hierarchical shape-by-shape assembly of virus-laden particles into larger ones to transfect two different genes on the seeded cells. We show that smaller daughter particles with different sizes and shapes can be assembled into the matching indentations of larger parent particles with different sizes and shapes. Then, we transfected a population of cells with two different gene-transfecting viruses, each of which was laden on the parent or daughter particles.

ELIPatch, a thumbnail-size patch with immunospot array for multiplexed protein detection from human skin surface.

Proteins secreted by skin have great potential as biomarkers for interpreting skin conditions. However, inconvenience in handling and bulky size of existing methods are existing limitations. Here, we describe a thumb-nail sized patch with the array of microdisks which captures multiple proteins from the skin surface. Microdisks with antibody on the surface enable multiplexed immunoassay. By self-assembly, microdisks are placed into 2-dimensional arrays on adhesive tape. The proposed Enzyme-Linked Immunospot array on a Patch shows sufficient sensitivity for IL-1α, IL1RA, IL-17A, IFN-g, and TNF-α, while IL-6 and IL-1ß are non-detectable in some cases. As demonstrations, we quantified cytokines from different skin regions and volunteers in a high-spatial-resolution.