Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity.

For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.

Correction to: L_RNA_scaffolder: scaffolding genomes with transcripts.

Following the publication of this article [1], the authors reported that the link to the software described in the article is no longer valid.

Transcriptome analysis between invasive Pomacea canaliculata and indigenous Cipangopaludina cahayensis reveals genomic divergence and diagnostic microsatellite/SSR markers.

BACKGROUND: Pomacea canaliculata is an important invasive species worldwide. However, little is known about the molecular mechanisms behind species displacement, adaptational abilities, and pesticide resistance, partly because of the lack of genomic information that is available for this species. Here, the transcriptome sequences for the invasive golden apple snail P. canaliculata and the native mudsnail Cipangopaludina cahayensis were obtained by next-generation-sequencing and used to compare genomic divergence and identify molecular markers. RESULTS: More than 46 million high quality sequencing reads were generated from P. canaliculata and C. cahayensis using Illumina paired-end sequencing technology. Our analysis indicated that 11,312 unigenes from P. canaliculata and C. cahayensis showed significant similarities to known proteins families, among which a total of 4,320 specific protein families were identified. KEGG pathway enrichment was analyzed for the unique unigenes with 17 pathways (p-value < 10(-5)) in P. canaliculata relating predominantly to lysosomes and vitamin digestion and absorption, and with 12 identified in C. cahayensis, including cancer and toxoplasmosis pathways, respectively. Our analysis also indicated that the comparatively high number of P450 genes in the P. canaliculata transcriptome may be associated with the pesticide resistance in this species. Additionally, 16,717 simple sequence repeats derived from expressed sequence tags (EST-SSRs) were identified from the 14,722 unigenes in P. canaliculata and 100 of them were examined by PCR, revealing a species-specific molecular marker that could distinguish between the morphologically similar P. canaliculata and C. cahayensis snails. CONCLUSIONS: Here, we present the genomic resources of P. canaliculata and C. cahayensis. Differentially expressed genes in the transcriptome of P. canaliculata compared with C. cahayensis corresponded to critical metabolic pathways, and genes specifically related to environmental stress response were detected. The CYP4 family of P450 cytochromes that may be important factors in pesticide metabolism in P. canaliculata was identified. Overall, these findings will provide valuable genetic data for the further characterization of the molecular mechanisms that support the invasive and adaptive abilities of P. canaliculata.

The transcriptomes of the crucian carp complex (Carassius auratus) provide insights into the distinction between unisexual triploids and sexual diploids.

Both sexual reproduction and unisexual reproduction are adaptive strategies for species survival and evolution. Unisexual animals have originated largely by hybridization, which tends to elevate their heterozygosity. However, the extent of genetic diversity resulting from hybridization and the genomic differences that determine the type of reproduction are poorly understood. In Carassius auratus, sexual diploids and unisexual triploids coexist. These two forms are similar morphologically but differ markedly in their modes of reproduction. Investigation of their genomic differences will be useful to study genome diversity and the development of reproductive mode. We generated transcriptomes for the unisexual and sexual populations. Genes were identified using homology searches and an ab initio method. Estimation of the synonymous substitution rate in the orthologous pairs indicated that the hybridization of gibel carp occurred 2.2 million years ago. Microsatellite genotyping in each individual from the gibel carp population indicated that most gibel carp genes were not tri-allelic. Molecular function and pathway comparisons suggested few gene expansions between them, except for the progesterone-mediated oocyte maturation pathway, which is enriched in gibel carp. Differential expression analysis identified highly expressed genes in gibel carp. The transcriptomes provide information on genetic diversity and genomic differences, which should assist future studies in functional genomics.

L_RNA_scaffolder: scaffolding genomes with transcripts.

BACKGROUND: Generation of large mate-pair libraries is necessary for de novo genome assembly but the procedure is complex and time-consuming. Furthermore, in some complex genomes, it is hard to increase the N50 length even with large mate-pair libraries, which leads to low transcript coverage. Thus, it is necessary to develop other simple scaffolding approaches, to at least solve the elongation of transcribed fragments. RESULTS: We describe L_RNA_scaffolder, a novel genome scaffolding method that uses long transcriptome reads to order, orient and combine genomic fragments into larger sequences. To demonstrate the accuracy of the method, the zebrafish genome was scaffolded. With expanded human transcriptome data, the N50 of human genome was doubled and L_RNA_scaffolder out-performed most scaffolding results by existing scaffolders which employ mate-pair libraries. In these two examples, the transcript coverage was almost complete, especially for long transcripts. We applied L_RNA_scaffolder to the highly polymorphic pearl oyster draft genome and the gene model length significantly increased. CONCLUSIONS: The simplicity and high-throughput of RNA-seq data makes this approach suitable for genome scaffolding. L_RNA_scaffolder is available at http://www.fishbrowser.org/software/L_RNA_scaffolder.

Trmt112 gene expression in mouse embryonic development.

Mouse Trmt112, the homologous gene of yeast Trm112 (tRNA methyltransferase 11-2), was initially cloned from RIKEN with uncertain function. The yeast TRM112 is now known to play important roles in RNA methylation. Here, we studied the expression of Trmt112 by in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). A higher expression level of Trmt112 was observed in the brain and nervous system by whole mount in situ hybridization from embryonic day 10.5 (E10.5) to E11.5. At later developmental stages E13.5 and E16.5, abundant expression was prominently found in various organs and tissues including developing brain, nervous system, thymus, lung, liver, intestine, kidney, and cartilage. Furthermore, Trmt112 was persistently expressed from E9.5 to E18.5 on whole embryos and highly expressed in multiple organs at E12.5, E15.5 and E18.5 by QRT-PCR. These results showed that Trmt112 gene was highly and ubiquitously expressed during mouse embryonic development, implying that it might be involved in the morphogenesis of diverse organs and tissues and numerous physiological functions.

Implications of mutational spectrum in myelodysplastic syndromes based on targeted next-generation sequencing.

Myelodysplastic syndromes (MDS) are a group of myeloid hematological malignancies, with a high risk of progression to acute myeloid leukemia (AML). To explore the role of acquired mutations in MDS, 111 MDS-associated genes were screened using next-generation sequencing (NGS), in 125 patients. One or more mutations were detected in 84% of the patients. Some gene mutations are specific for MDS and were associated with disease subtypes, and the patterns of mutational pathways could be associated with progressive MDS. The patterns, frequencies and functional pathways of gene mutations are different, but somehow related, between MDS and AML. Multivariate analysis suggested that patients with ≥ 2 mutations had poor progression-free survival, while GATA1/GATA2, DNMT3A and KRAS/NRAS mutations were associated with poor overall survival. Based on a novel system combining IPSS-R and molecular markers, these MDS patients were further divided into 3 more accurate prognostic subgroups. A panel of 11 target genes was proposed for genetic profiling of MDS. The study offers new insights into the molecular signatures of MDS and the genetic consistency between MDS and AML. Furthermore, results indicate that MDS could be classified by mutation combinations to guide the administration of individualized therapeutic interventions.

Mutational spectrum and risk stratification of intermediate-risk acute myeloid leukemia patients based on next-generation sequencing.

Intermediate-risk acute myeloid leukemia (IR-AML), which accounts for a substantial number of AML cases, is highly heterogeneous. Although several mutations have been identified, the heterogeneity of AML is uncertain because novel mutations have yet to be discovered. Here we applied next generation sequencing (NGS) platform to screen mutational hotspots in 410 genes relevant to hematological malignancy. IR-AML samples (N=95) were sequenced by Illumina Hiseq and mutations in 101 genes were identified. Only seven genes (CEBPA, NPM1, DNMT3A, FLT3-ITD, NRAS, IDH2 and WT1) were mutated in more than 10% of patients. Genetic interaction analysis identified several cooperative and exclusive patterns of overlapping mutations. Mutational analysis indicated some correlation between genotype and phenotype. FLT3-ITD mutations were identified as independent factors of poor prognosis, while CEBPA mutations were independent favorable factors. Co-occurrence of FLT3-ITD, NPM1 and DNMT3A mutations was identified with associated with specific clinical AML features and poor outcomes. Furthermore, by integrating multiple mutations in the survival analysis, 95 IR-AML patients could be stratified into three distinct risk groups allowing reductions in IR-AML by one-third. Our study offers deep insights into the molecular pathogenesis and biology of AML and indicated that the prognosis of IR-AML could be further stratified by different mutation combinations which may direct future treatment intervention.

Complete mitochondrial genome of the kissing gourami, Helostoma temminkii.

The kissing gourami (Helostoma temminkii) belongs to the Labyrinth fishes (Perciformes: Anabantoidei), which exhibits a wide variety of behavioral traits. In this study the complete mitogenome of H. temminkii was determined to be 16,740 bp in length. It contains 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The sequence structure of non-coding control region was also analyzed. Comparing the mitochondrial genome of H. temminkii with its closely related species Colisa lalia showed the similarity of 78%. The complete mitochondrial genome of H. temminkii provides resource for phylogenetic analysis on Anabantoidei.

The fate of recent duplicated genes following a fourth-round whole genome duplication in a tetraploid fish, common carp (Cyprinus carpio).

Whole genome duplication (WGD) results in extensive genetic redundancy. In plants and yeast, WGD is followed by rapid gene deletions and intense expression differentiation with slow functional divergence. However, the early evolution of the gene differentiation processes is poorly understood in vertebrates because almost all studied WGDs are extremely ancient, and the genomes have returned to a diploid status. Common carp had a very recent fourth round of WGD dated to 8 million years ago. It therefore constitutes an ideal model to study early-stage functional divergence and expression differentiation in vertebrates. We identified 1,757 pairs of recently duplicated genes (RDGs) originating from this specific WGD and found that most ancestral genes were retained in duplicate. Most RDGs were conserved and under selective pressure. Gene expression analysis across six tissues revealed that 92.5% of RDG pairs were co-expressed in at least one tissue and that the expression of nearly half pairs ceased to be strongly correlated, indicating slow spatial divergence but rapid expression dissociation. Functional comparison revealed that 25% of pairs had functional divergence, of which neo- and sub-functionalization were the main outcomes. Our analysis revealed slow gene loss but rapid and intense expression and function differentiation after WGD.

Genome sequence and genetic diversity of the common carp, Cyprinus carpio.

The common carp, Cyprinus carpio, is one of the most important cyprinid species and globally accounts for 10% of freshwater aquaculture production. Here we present a draft genome of domesticated C. carpio (strain Songpu), whose current assembly contains 52,610 protein-coding genes and approximately 92.3% coverage of its paleotetraploidized genome (2n = 100). The latest round of whole-genome duplication has been estimated to have occurred approximately 8.2 million years ago. Genome resequencing of 33 representative individuals from worldwide populations demonstrates a single origin for C. carpio in 2 subspecies (C. carpio Haematopterus and C. carpio carpio). Integrative genomic and transcriptomic analyses were used to identify loci potentially associated with traits including scaling patterns and skin color. In combination with the high-resolution genetic map, the draft genome paves the way for better molecular studies and improved genome-assisted breeding of C. carpio and other closely related species.

Complete mitochondrial genome of Chinese sleeper, Perccottus glenii.

Chinese sleeper (Perccottus glenii) belongs to the largest vertebrate order, Perciformes. In this study, the complete mitochondrial genome of P. glenii was determined to be 16,487 bp in length, including 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. We also analysed the sequence structure of non-coding control region. Comparing the mitochondrial genome of P. glenii with its congener Rhyacichthys aspro showed sequence similarity and the identical gene arrangement. The complete mitochondrial genome of Chinese sleeper provides the basis for the studies in Perciformes evolution and conservation.

A dense genetic linkage map for common carp and its integration with a BAC-based physical map.

BACKGROUND: Common carp (Cyprinus carpio) is one of the most important aquaculture species with an annual global production of 3.4 million metric tons. It is also an important ornamental species as well as an important model species for aquaculture research. To improve the economically important traits of this fish, a number of genomic resources and genetic tools have been developed, including several genetic maps and a bacterial artificial chromosome (BAC)-based physical map. However, integrated genetic and physical maps are not available to study quantitative trait loci (QTL) and assist with fine mapping, positional cloning and whole genome sequencing and assembly. The objective of this study was to integrate the currently available BAC-based physical and genetic maps. RESULTS: The genetic map was updated with 592 novel markers, including 312 BAC-anchored microsatellites and 130 SNP markers, and contained 1,209 genetic markers on 50 linkage groups, spanning 3,565.9 cM in the common carp genome. An integrated genetic and physical map of the common carp genome was then constructed, which was composed of 463 physical map contigs and 88 single BACs. Combined lengths of the contigs and single BACs covered a physical length of 498.75 Mb, or around 30% of the common carp genome. Comparative analysis between common carp and zebrafish genomes was performed based on the integrated map, providing more insights into the common carp specific whole genome duplication and segmental rearrangements in the genome. CONCLUSION: We integrated a BAC-based physical map to a genetic linkage map of common carp by anchoring BAC-associated genetic markers. The density of the genetic linkage map was significantly increased. The integrated map provides a tool for both genetic and genomic studies of common carp, which will help us to understand the genomic architecture of common carp and facilitate fine mapping and positional cloning of economically important traits for genetic improvement and modification.