Parallel DNA Research Articles

Interactions between PHD3-Bromo of MLL1 and H3K4me3 Revealed by Single-Molecule Magnetic Tweezers in a Parallel DNA Circuit.

Single-molecule force spectroscopy is a powerful tool to directly measure protein-protein interactions (PPI). The high specificity and precision of PPI measurements made it possible to reveal detailed mechanisms of intermolecular interactions. However, protein aggregation due to specific or nonspecific interactions is among the most challenging problems in PPI examination. Here, we propose a strategy of a parallel DNA circuit to probe PPI using single-molecule magnetic tweezers. In contrast to PPI examination using atomic force microscopy, microspheres as probes used in magnetic tweezers avoided the single-probe issue of a cantilever. Negatively charged DNA as a linker circumvented the severe aggregation in the PPI construct with a protein linker. The unnatural amino acid encoded in proteins of interest expanded the choices of biorthogonal conjugation. We demonstrated how to apply our strategy to probe the PPI between the PHD3-Bromo and the histone H3 methylated at K4, a critical epigenetic event in leukemia development. We found a rupture force of 12 pN for breaking the PPI, which is much higher than that required to peel DNA off from a nucleosome, 3 pN. We expect that our methods will make PPI measurements of mechanics and kinetics with great precision, facilitating PPI-related research, e.g., PPI-targeted drug discovery.

Substituent dependent selectivity of fluorescent chemosensors derived from coumarin for biologically relevant DNA structures and anions

In this study, we describe facile synthesis of a series of coumarin appended Schiff base chemosensors for the detection of spesific anions such as F−, CN−, AcO−, and H2PO4−. The sensors have been also employed for signaling singlestranded, doublestranded, as well as parallel, anti-parallel and hybrid G-quadruplex DNA structures and increased specificity was detected towards CN− and parallel G-quadruplex structure. The substituents and their positions (ortho, meta, or para) in relation to the OH impacted significantly on the sensing of the anions as well as the acidity of the OH group, in the recognition of the anions. The deprotonation and addition sensing mechanisms of the probes were investigated through UV–vis, fluorimetric, and NMR titration methods. The two signalling mechanisms also have been confirmed using DFT calculations. The bias studies of all the compounds indicated an immediate visible fluorescence change when CN was added to a partial aqueous mixture of DMSO/tap water (6:4, v/v). Furthermore, the chemosensor were studied in neutral and basic mixtures of DMSO/buffer (6:4, v/v) for absorption and emission spectra in the presence and absence of CN−. In the case of DNA structures, up to 5.5-fold fluorescence enhancement was observed in the presence of parallel G-quadruplex as compared with only 1.3-fold in the presence of duplex DNA. Molecular docking studies revealed that, the selectivity is as a result of increased number of H-bonds between DNA and 2b due to modified substituent. In a nutshell, all the fluorophores can be employed in sensing CN− in partial aqueous solution, while 2a, 2b, 2e and 2f showed strong selectivity towards parallel G-quadruplexes over other DNA topologies. Thermal studies of the compounds have shown that the compounds have enough thermal stability properties for application as optic dye.

Optomagnetic Detection of Rolling Circle Amplification Products.

Rolling circle amplification (RCA) of a synthetic nucleic acid target is detected using magnetic nanoparticles (MNPs) combined with an optomagnetic (OM) readout. Two RCA assays are developed with on-chip detection of rolling circle products (RCPs) either at end-point where MNPs are mixed with the sample after completion of RCA or in real time where MNPs are mixed with the sample during RCA. The plastic chip acts as a cuvette, which is positioned in a setup integrated with temperature control and simultaneous detection of four parallel DNA hybridization reactions between functionalized MNPs and products of DNA amplification. The OM technique probes the small-angle rotation of MNPs bearing oligonucleotide probes complementary to the repeated nucleotide sequence of the RCPs. This rotation is restricted when MNPs bind to RCPs, which can be observed as a turn-off of the signal from MNPs that are free to rotate. The amount of MNPs bound to RCPs is found to increase in response to the amplification time as well as in response to the synthetic DNA target concentration (2-40pM dynamic range). We report OM real-time results obtained with MNPs present during RCA and compare to relevant end-point OM results for RCPs generated for different RCA times. The real-time approach avoids opening of tubes post-RCA and thus reduces risk of lab contamination with amplification products without compromising the sensitivity and dynamic range of the assay.

Decoding regulatory structures and features from epigenomics profiles: A Roadmap-ENCODE Variational Auto-Encoder (RE-VAE) model.

The development of chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing (ChIP-seq) technologies has promoted generation of large-scale epigenomics data, providing us unprecedented opportunities to explore the landscape of epigenomic profiles at scales across both histone marks and tissue types. In addition to many tools directly for data analysis, advanced computational approaches, such as deep learning, have recently become promising to deeply mine the data structures and identify important regulators from complex functional genomics data. We implemented a neural network framework, a Variational Auto-Encoder (VAE) model, to explore the epigenomic data from the Roadmap Epigenomics Project and the Encyclopedia of DNA Elements (ENCODE) project. Our model is applied to 935 reference samples, covering 28 tissues and 12 histone marks. We used the enhancer and promoter regions as the annotation features and ChIP-seq signal values in these regions as the feature values. Through a parameter sweep process, we identified the suitable hyperparameter values and built a VAE model to represent the epigenomics data and to further explore the biological regulation. The resultant Roadmap-ENCODE VAE (RE-VAE) model contained data compression and feature representation. Using the compressed data in the latent space, we found that the majority of histone marks were well clustered but not for tissues or cell types. Tissue or cell specificity was observed only in some histone marks (e.g., H3K4me3 and H3K27ac) and could be characterized when the number of tissue samples is large (e.g., blood and brain). In blood, the contributive regions and genes identified by RE-VAE model were confirmed by tissue-specificity enrichment analysis with an independent tissue expression panel. Finally, we demonstrated that RE-VAE model could detect cancer cell lines with similar epigenomics profiles. In conclusion, we introduced and implemented a VAE model to represent large-scale epigenomics data. The model could be used to explore classifications of histone modifications and tissue/cell specificity and to classify new data with unknown sources.

Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext).

Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

981P - TERT gene fusions characterize a subset of metastatic Leydig cell tumours

BackgroundMetastatic Leydig cells tumours (LCT) are rare, difficult to treat malignancies without known underlying molecular-genetic events. An index case of metastatic LCT showed LDLR-TERT gene fusion upon routine genetic profiling for detection of therapeutic targets, which was then followed by an investigation into a cohort of additional LCT. Methods20 LCT (7 metastatic and 13 primary tumours) from 3 participating institutions (Caris Life Sciences, Phoenix, USA; Clinical Hospital Center, Zagreb, Croatia and Biopticka Laborator, Plzen, Czech Republic) were analyzed using massively parallel DNA and RNA sequencing (NGS) approach. Immunohistochemistry (IHC) was used for detection of selected protein biomarkers. ResultsTERT gene fusions were detected only in metastatic Leydig cell tumours, in three of 5 successfully analyzed cases (RMST:TERT, LDLR:TERT and B4GALT5:TERT, respectively). The case with B4GALT5:TERT fusion also showed amplifications (>6 copies) of TOP1 and CCND3 genes. TP53 mutation (M246I) was detected in one metastatic tumour without TERT fusion. No gene fusions were found in any primary tumours (0/8 successfully analyzed). 3/6 primary tumours showed multiple gene amplifications, without a consistent pattern. At the protein level (IHC) 5/7 metastatic and 6/7 primary tumours over-expressed TOP1. Full length androgen receptor (AR) was overexpressed in 7/8 primary tumours (without detectable ARv7 mRNA or ARv7 protein), but only in 1 of 5 metastatic LCT. No tumours exhibited high tumour mutational burden, microsatellite instability nor expressed PD-L1 in tumour cells. ConclusionsOur study for the first time identified TERT gene fusions as a main/sole detected genetic alteration and a potential therapeutic target in malignant, metastatic Leydig cell tumours. Additional biomarkers (TOP1 amplification and over-expression) may help guide decisions on chemotherapy for selected individual patients. Androgen receptor blockade may be considered in AR overexpressing tumours without evidence of ARv7 presence. Legal entity responsible for the studyCaris Life Sciences. FundingCaris Life Sciences. DisclosureZ. Gatalica: Leadership role: Caris Life Sciences. J. Xiu: Leadership role, employment: Caris Life Sciences. J. Swensen: Leadership role, employment: Caris Life Sciences. All other authors have declared no conflicts of interest.

P2570Synergistic activation of the cardiac enhancer landscape during reprogramming

Abstract Introduction Cardiogenic transcription factors (TFs), Gata4, Mef2c, and Tbx5, can directly reprogram fibroblasts to a cardiac fate and their cardiogenic activity is enhanced by the Hand2 TF and the Akt1 kinase. Although these cardiac reprogramming factors are key regulators of heart development, their expression and biological functions are not limited to the heart, and the mechanism by which these cardiac factors orchestrate reprogramming remains unclear. Purpose We sought to study the molecular mechanisms by which cardiac reprogramming factors contribute to cell fate conversion using a genome-wide approach. Methods Here, we used chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) to explore the genomic binding sites of reprogramming TFs and the landscape of active enhancers, annotated by H3K27ac histone modification, during cardiac reprogramming. Results We found that reprogramming TFs rapidly silence fibroblast enhancers and synergistically activate cardiac enhancers which were predominantly enriched with Mef2 motifs. Addition of Hand2 and Akt1 to GMT expands TF co-occupancy and thereby activates additional cardiac enhancers, which further augments cardiac gene expression. Moreover, additional cardiac enhancers were sequentially activated during the reprogramming process, in accordance with the temporal acquisition of functional phenotypes in induced cardiac-like myocytes. We discovered that subsets of reprogramming enhancers are conserved among other cardiogenic processes, and a collection of reprogramming enhancers displayed unique spatial expression patterns in the developing heart in transgenic mouse embryos. Conclusion Our study describes the epigenomic dynamics that underlie cardiac reprogramming which is cooperatively orchestrated by reprogramming factors to convert fibroblasts toward a cardiac lineage. Acknowledgement/Funding NIH (AR-067294, HL-130253, HL-138426, HD-087351), Fondation Leducq Transatlantic Networks of Excellence in Cardiovascular Research

Genomic and Epigenomic Features of Primary and Recurrent Hepatocellular Carcinomas

Intratumor heterogeneity and divergent clonal lineages within and among primary and recurrent hepatocellular carcinomas (HCCs) produce challenges to patient management. We investigated genetic and epigenetic variations within liver tumors, among hepatic lesions, and between primary and relapsing tumors. Tumor and matched nontumor liver specimens were collected from 113 patients who underwent partial hepatectomy for primary or recurrent HCC at 2 hospitals in Hong Kong. We performed whole-genome, whole-exome, or targeted capture sequencing analyses of 356 HCC specimens collected from multiple tumor regions and matched initial and recurrent tumors. We performed parallel DNA methylation profiling analyses of 95 specimens. Genomes and epigenomes of nontumor tissues that contained areas of cirrhosis or fibrosis were analyzed. We developed liver cancer cell lines that endogenously expressed a mutant form of TP53 (R249S) or overexpressed mutant forms of STAT3 (D170Y, K348E, and Y640F) or JAK1 (S703I and L910P) and tested the abilities of pharmacologic agents to reduce activity. Cells were analyzed by immunoblotting and chromatin immunoprecipitation with quantitative polymerase chain reaction. We determined the monoclonal origins of individual tumors using a single sample collection approach that captured more than 90% of mutations that are detected in all regions of tumors. Phylogenetic and phylo-epigenetic analyses revealed interactions and codependence between the genomic and epigenomic features of HCCs. Methylation analysis revealed a field effect in cirrhotic liver tissues that predisposes them to tumor development. Comparisons of genetic features revealed that 52% of recurrent HCCs derive from the clonal lineage of the initial tumor. The clonal origin if recurrent HCCs allowed construction of a temporal map of genetic alterations that associated with tumor recurrence. Activation of JAK signaling to STAT was a characteristic of HCC progression via mutations that associate with response to drug sensitivity. The combination of a mutation that increases the function of TP53 and the 17p chromosome deletion might provide liver cancer cells with a replicative advantage. Chromatin immunoprecipitation analysis of TP53 with the R249S substitution revealed its interaction with genes that encode chromatin regulators (MLL1 and MLL2). We validated MLL1 and MLL2 as direct targets of TP53R249S and affirmed their association in the Cancer Genome Atlas dataset. The MLL-complex antagonists MI-2-2 (inhibitor of protein interaction) and OICR-9492 (inhibitor of activity) specifically inhibited proliferation of HCC cells that express TP53R249S at nanomolar concentrations. We performed a systematic evaluation of intra- and intertumor genetic heterogeneity in HCC samples and identified genetic and epigenetic changes that associate with tumor progression and recurrence. We identified chromatin regulators that are upregulated by mutant TP53 in HCC cells and inhibitors that reduce proliferation of these cells. DNA methylation patterns in cirrhotic or fibrotic liver tissues might be used to identify those at risk of HCC development.

Mutational Spectrum and Clinical Features of Patients with LOXHD1 Variants Identified in an 8074 Hearing Loss Patient Cohort.

Variants of the LOXHD1 gene, which are expressed in hair cells of the cochlea and vestibule, have been reported to cause a progressive form of autosomal recessive non-syndromic hereditary hearing loss, DFNB77. In this study, genetic screening was conducted on 8074 Japanese hearing loss patients utilizing massively parallel DNA sequencing to identify individuals with LOXHD1 variants and to assess their phenotypes. A total of 28 affected individuals and 21 LOXHD1 variants were identified, among which 13 were novel variants. A recurrent variant c.4212 + 1G > A, only reported in Japanese patients, was detected in 18 individuals. Haplotype analysis implied that this variation occurred in a mutational hot spot, and that multiple ancestors of Japanese population had this variation. Patients with LOXHD1 variations mostly showed early onset hearing loss and presented different progression rates. We speculated that the varying severities and progression rates of hearing loss are the result of environmental and/or other genetic factors. No accompanying symptoms, including vestibular dysfunction, with hearing loss were detected in this study. Few studies have reported the clinical features of LOXHD1-gene associated hearing loss, and this study is by far the largest study focused on the evaluation of this gene.

Identification of new hypoxia-regulated epithelial-mesenchymal transition marker genes labeled by H3K4 acetylation.

Hypoxia-induced epithelial-mesenchymal transition (EMT) involves the interplay between chromatin modifiers histone deacetylase 3 (HDAC3) and WDR5. The histone mark histone 3 lysine 4 acetylation (H3K4Ac) is observed in the promoter regions of various EMT marker genes (eg, CDH1 and VIM). To further define the genome-wide location of H3K4Ac, a chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) analysis was performed using a head and neck squamous cell carcinoma (HNSCC) FaDu cell line under normoxia and hypoxia. H3K4Ac was found to be located mainly around the transcription start site. Coupled with analysis of gene expression by RNA sequencing and using a HDAC3 knockdown cell line, 10 new genes (BMI1, GLI1, SMO, FOXF1, SIRT2, etc) that were labeled by H3K4Ac and regulated by HDAC3 were identified. Overexpression or knockdown of GLI1/SMO increased or repressed the in vitro migration and invasion activity in OECM-1/FaDu cells, respectively. In HNSCC patients, coexpression of GLI1 and SMO in primary tumors correlated with metastasis. Our results identify new EMT marker genes that may play a significant role in hypoxia-induced EMT and metastasis and further provide diagnostic and prognostic implications.

Parallel Serial Assessment of Somatic Mutation and Methylation Profile from Circulating Tumor DNA Predicts Treatment Response and Impending Disease Progression in Osimertinib-Treated Lung Adenocarcinoma Patients

Background: Circulating tumor DNA (ctDNA) harboring tumor-specific genetic and epigenetic aberrations allows for early detection and real-time monitoring of tumor dynamics. In this study, we aimed to evaluate the potential of parallel serial assessment of somatic mutation and methylation profile in monitoring the response to osimertinib of EGFR T790M-positive advanced lung adenocarcinoma patients. Methods: Parallel somatic mutation and DNA methylation profiling was performed on a total of 85 longitudinal plasma samples obtained from eight stage IV osimertinib-treated EGFR T790M-positive lung adenocarcinoma patients. Findings: Our results revealed significant correlation between the by-patient methylation level with the maximum allele fraction (maxAF, P=0.0002). The methylation levels were significantly higher in the plasma samples of patients with detectable somatic mutations than patients without somatic mutations (P=0.0003) and healthy controls (P=0.0018). Moreover, analysis of both DNA methylation level and maxAF revealed four trends of treatment response. Collectively, the decrease in methylation level and maxAF reflected treatment efficacy, while the gradual increase reflected impending disease progression. Elevated methylation levels and maxAF were observed in six and five patients in an average lead-time of 3.0 and 1.9 months, respectively, prior to evaluation of disease progression using radiological imaging. Interpretation: DNA methylation profiling has the potential to predict disease relapse prior to being evaluated thru radiological modalities, suggesting that serial assessment of methylation level in combination with somatic mutation profiling are reliable methods for treatment monitoring and should be incorporated with imaging modalities for a more comprehensive work-up of treatment response, particularly for patients treated with targeted therapies. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (grant number 81772471 to S.Xia), Double First-Class University Plan funding of 2016 from Tongji Medical College, Huazhong University of Science and Technology (grant number 5001540022 to S.Xia) and Qinghai Science and Technology Department Funding (grant number 2017-ZJ-709 to S. Xia). Declaration of Interests: J.Ye, A.Lizaso, J.Su, H.Han-Zhang and S. Chuai are employees of Burning Rock Biotech. The other authors declare no potential conflicts of interest. Ethics Approval Statement: This study was performed according to the Helsinki Declaration of 1964 and its current amendments. The study protocol has been approved by the Ethics Committee of Huazhong University of Science and Technology (approval number: 2015L01374/2015L01373). Written informed consent was provided by all the patients included in the study.

Barcoded DNA nanostructures for the multiplexed profiling of subcellular protein distribution.

Massively parallel DNA sequencing is established, yet high-throughput protein profiling remains challenging. Here, we report a barcoding approach that leverages the combinatorial sequence content and the configurational programmability of DNA nanostructures for high-throughput multiplexed profiling of the subcellular expression and distribution of proteins in whole cells. The barcodes are formed by in situ hybridization of tetrahedral DNA nanostructures and short DNA sequences conjugated with protein-targeting antibodies, and by nanostructure-assisted ligation (either enzymatic or chemical) of the nanostructures and exogenous DNA sequences bound to nanoparticles of different sizes (which cause these localization sequences to differentially distribute across subcellular compartments). Compared with linear DNA barcoding, the nanostructured barcodes enhance the signal by more than 100-fold. By implementing the barcoding approach on a microfluidic device for the analysis of rare patient samples, we show that molecular subtypes of breast cancer can be accurately classified and that subcellular spatial markers of disease aggressiveness can be identified.

Silver(I)-mediated base pairing in parallel-stranded DNA involving the luminescent cytosine analog 1,3-diaza-2-oxophenoxazine.

1,3-Diaza-2-oxophenoxazine (X) has been introduced as a ligand in silver(I)-mediated base pairing in a parallel DNA duplex. This fluorescent cytosine analog is capable of forming stabilizing X-Ag(I)-X and X-Ag(I)-C base pairs in DNA duplexes, as confirmed by temperature-dependent UV spectroscopy and luminescence spectroscopy. DFT calculations of the silver(I)-mediated base pairs suggest the presence of a synergistic hydrogen bond. Molecular dynamics (MD) simulations of entire DNA duplexes nicely underline the geometrical flexibility of these base pairs, with the synergistic hydrogen bond facing either the major or the minor groove. Upon silver(I) binding to the X:X or X:C base pairs, the luminescence emission maximum experiences a red shift from 448 to 460nm upon excitation at 370nm. Importantly, the luminescence of the 1,3-diaza-2-oxophenoxazine ligand is not quenched significantly upon binding a silver(I) ion. In fact, the luminescence intensity even increases upon formation of a X-Ag(I)-C base pair, which is expected to be beneficial for the development of biosensors. As a consequence, the silver(I)-mediated phenoxazinone base pairs represent the first strongly fluorescent metal-mediated base pairs.

Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening.

Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3' end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.

Generation of Highly Biomimetic Quality Control Materials for Noninvasive Prenatal Testing Based on Enzymatic Digestion of Matched Mother-Child Cell Lines.

Noninvasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) is widely used. However, biomimetic quality control materials that have properties identical to clinical samples and that are applicable to a wide range of methodologies are still not available to support assay development, internal quality control, and proficiency testing. We developed a set of dual enzyme-digested NIPT quality control materials (DENQCMs) that comprise simulated human plasma and mixtures of mother cell line-derived cfDNA based on DNA fragmentation factor digestion (D-cfDNA) and the matched child cell line-derived cfDNA based on micrococcal nuclease digestion (M-cfDNA). Serially diluted samples positive for trisomies 21, 18, and 13 were included in the materials. To evaluate the biomimetics, DENQCMs were analyzed using random massively parallel sequencing (MPS), targeted MPS, and imaging single DNA molecule methods, and the estimated fetal fractions (FFs) were compared with expected FFs. Genome-wide analysis of cfDNA fragmentation patterns was performed to confirm their biological characteristics. The genetic status of each DENQCM was correctly detected by 4 routine NIPT assays for the samples with FFs >5%. The chromosome Y-based and single-nucleotide polymorphism-based estimations of FFs were linearly related to those expected FFs. The MPS results exhibited a concordance of quality metrics between DENQCMs and maternal plasma, such as GC contents of cfDNA and unique read ratios. The DENQCMs are universally applicable for different platforms. We propose DENQCMs as an approach to produce matched maternal and fetal cfDNA that will be suitable for the preparation of quality control materials for NIPT.

An Efficient Technique for Double Faults Detection and their Locations Identification in Digital Microfluidic Biochip

Progress of digital microfluidic biochip (DMFB) confronts for the defective and specious electrodes. Not only these hinder the routing of droplets but also the completion time of assay is influenced by those defective electrodes. As Microfluidic-based biochips are broadly used in the revolution of medical diagnosis, gigantic parallel DNA analysis, automatic drug discovery and real-time biomolecular recognition including numerous safety-critical applications, this biochip definitely responsible for appropriate and accurate result. Prior accepting it for perceptive purposes the microfluidic biochip must confirm its precision and robustness. In this article, an aspect of fast fault diagnosis appliance for perceiving double faults and recognizing the fault locations within the biochip is introduced. If the biochip is defect free then the proposed approach computes the traversal time as well. The suggested result outpoured that the propound technique is competent, efficacious as well as delineate signifying improvement over the surviving method. Furthermore this paper added expedient reconfiguration contrivance.

Arabidopsis histone H3K4 demethylase JMJ17 functions in dehydration stress response.

Under dehydration in plants, antagonistic activities of histone 3 lysine 4 (H3K4) methyltransferase and histone demethylase maintain a dynamic and homeostatic state of gene expression by orientating transcriptional reprogramming toward growth or stress tolerance. However, the histone demethylase that specifically controls histone methylation homeostasis under dehydration stress remains unknown. Here, we document that a histone demethylase, JMJ17, belonging to the KDM5/JARID1 family, plays crucial roles in response to dehydration stress and abscisic acid (ABA) in Arabidopsis thaliana. jmj17 loss-of-function mutants displayed dehydration stress tolerance and ABA hypersensitivity in terms of stomatal closure. JMJ17 specifically demethylated H3K4me1/2/3 via conserved iron-binding amino acids invitro and invivo. Moreover, H3K4 demethylase activity of JMJ17 was required for dehydration stress response. Systematic combination of genome-wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) analyses revealed that a loss-of-function mutation in JMJ17 caused an ectopic increase in genome-wide H3K4me3 levels and activated a plethora of dehydration stress-responsive genes. Importantly, JMJ17 bound directly to the chromatin of OPEN STOMATA 1 (OST1) and demethylated H3K4me3 for the regulation of OST1 mRNA abundance, thereby modulating the dehydration stress response. Our results demonstrate a new function of a histone demethylase under dehydration stress in plants.

Validation of a comprehensive cancer genomic profiling assay based on massively parallel DNA sequencing.

e13138 Background: Validation is very important for diagnostic assays, here we describe a validation process of a comprehensive cancer genomic profiling assay based on massively parallel DNA sequencing. This assay can detect base substitutions, short insertions and deletions, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI) across 543 cancer-related genes from tumor specimens or plasma. Methods: Analytical validation was conducted with 20 cell line pools whose mutation were verified by digital droplet PCR (ddPCR). Clinical validation was conducted with Proficiency Test samples and clinical samples whose mutation were verified by ddPCR. Sensitivity, positive predictive value (PPV) and precision of tumor specimens and plasma were assessed across the reportable range of the assay. For measurement of TMB， NGS libraries of a cohort of tumor samples were tested by whole exosome panel (WES) and this assay respectively. And clinical tumor tissue samples whose MSI status were identified by fluorescent PCR-capillary electrophoresis were also tested by this assay. Results: The SNV/Indel LOD95 was 0.6% for hot-spot variants, and 1.3% for panel-wised variants of tumor tissue samples with high specificity (PPV >99%). The SNV/Indel LOD95 was 0.4% for hot-spot variants, and 1.1% for panel-wised variants of plasma samples (cfDNA≥15ng), with PPV >99%. LOD95 of CNV was 2.2-2.3, with PPV>99%. Precision of CNV was higher than 95%, precision of SNV/Indel was higher than 97%. Concordance between TMB results tested by WES and this assay was 0.94. MSI results identified by this assay was the same as fluorescent PCR-capillary electrophoresis method. Conclusions: In summary, we present the analytical and clinical validation of a comprehensive NGS-based diagnostic assay for comprehensive tumor genomic profiling.

Next-Generation Sequencing and Emerging Technologies.

Genetic sequencing technologies are evolving at a rapid pace with major implications for research and clinical practice. In this review, the authors provide an updated overview of next-generation sequencing (NGS) and emerging methodologies. NGS has tremendously improved sequencing output while being more time and cost-efficient in comparison to Sanger sequencing. The authors describe short-read sequencing approaches, such as sequencing by synthesis, ion semiconductor sequencing, and nanoball sequencing. Third-generation long-read sequencing now promises to overcome many of the limitations of short-read sequencing, such as the ability to reliably resolve repeat sequences and large genomic rearrangements. By combining complementary methods with massively parallel DNA sequencing, a greater insight into the biological context of disease mechanisms is now possible. Emerging methodologies, such as advances in nanopore technology, in situ nucleic acid sequencing, and microscopy-based sequencing, will continue the rapid evolution of this area. These new technologies hold many potential applications for hematological disorders, with the promise of precision and personalized medical care in the future.

OTOF mutation analysis with massively parallel DNA sequencing in 2,265 Japanese sensorineural hearing loss patients.

The OTOF gene (Locus: DFNB9), encoding otoferlin, is reported to be one of the major causes of non-syndromic recessive sensorineural hearing loss, and is also reported to be the most common cause of non-syndromic recessive auditory neuropathy spectrum disorder (ANSD). In the present study, we performed OTOF mutation analysis using massively parallel DNA sequencing (MPS). The purpose of this study was to reveal the frequency and precise genetic and clinical background of OTOF-related hearing loss in a large hearing loss population. A total of 2,265 Japanese sensorineural hearing loss (SNHL) patients compatible with autosomal recessive inheritance (including sporadic cases) from 53 otorhinolaryngology departments nationwide participated in this study. The mutation analysis of 68 genes, including the OTOF gene, reported to cause non-syndromic hearing loss was performed using MPS. Thirty-nine out of the 2,265 patients (1.72%) carried homozygous or compound heterozygous mutations in the OTOF gene. It is assumed that the frequency of hearing loss associated with OTOF mutations is about 1.72% of autosomal recessive or sporadic SNHL cases. Hearing level information was available for 32 of 39 patients with biallelic OTOF mutations; 24 of them (75.0%) showed profound hearing loss, 7 (21.9%) showed severe hearing loss and 1 (3.1%) showed mild hearing loss. The hearing level of patients with biallelic OTOF mutations in this study was mostly severe to profound, which is consistent with the results of past reports. Eleven of the 39 patients with biallelic OTOF mutations had been diagnosed with ANSD. The genetic diagnosis of OTOF mutations has significant benefits in terms of clinical decision-making. Patients with OTOF mutations would be good candidates for cochlear implantation; therefore, the detection of OTOF mutations is quite beneficial for patients, especially for those with ANSD.