An Efficient Self-Organized Detection System for Algae.

Algal blooms have seriously affected the production and life of people and real-time detection of algae in water samples is a powerful measure to prevent algal blooms. The traditional manual detection of algae with a microscope is extremely time-consuming. In recent years, although there have been many studies using deep learning to classify and detect algae, most of them have focused on the relatively simple task of algal classification. In addition, some existing algal detection studies not only use small datasets containing limited algal species, but also only prove that object detection algorithms can be applied to algal detection tasks. These studies cannot implement the real-time detection of algae and timely warning of algal blooms. Therefore, this paper proposes an efficient self-organized detection system for algae. Benefiting from this system, we propose an interactive method to generate the algal detection dataset containing 28,329 images, 562,512 bounding boxes and 54 genera. Then, based on this dataset, we not only explore and compare the performance of 10 different versions of state-of-the-art object detection algorithms for algal detection, but also tune the detection system we built to its optimum state. In practical application, the system not only has good algal detection results, but also can complete the scanning, photographing and detection of a 2 cm × 2 cm, 0.1 mL algal slide specimen within five minutes (the resolution is 0.25886 µm/pixel); such a task requires a well-trained algal expert to work continuously for more than three hours. The efficient algal self-organized detection system we built makes it possible to detect algae in real time. In the future, with the help of IoT, we can use various smart sensors, actuators and intelligent controllers to achieve real-time collection and wireless transmission of algal data, use the efficient algal self-organized detection system we built to implement real-time algal detection and upload the detection results to the cloud to realize timely warning of algal blooms.

Evaluation of the Bacterial Diversity in the Human Tongue Coating Based on Genus-Specific Primers for 16S rRNA Sequencing.

The characteristics of tongue coating are very important symbols for disease diagnosis in traditional Chinese medicine (TCM) theory. As a habitat of oral microbiota, bacteria on the tongue dorsum have been proved to be the cause of many oral diseases. The high-throughput next-generation sequencing (NGS) platforms have been widely applied in the analysis of bacterial 16S rRNA gene. We developed a methodology based on genus-specific multiprimer amplification and ligation-based sequencing for microbiota analysis. In order to validate the efficiency of the approach, we thoroughly analyzed six tongue coating samples from lung cancer patients with different TCM types, and more than 600 genera of bacteria were detected by this platform. The results showed that ligation-based parallel sequencing combined with enzyme digestion and multiamplification could expand the effective length of sequencing reads and could be applied in the microbiota analysis.

Improving the microbial community reconstruction at the genus level by multiple 16S rRNA regions.

BACKGROUND: 16S rRNA genes have been widely used for phylogenetic reconstruction and the quantification of microbial diversity through the application of next-generation sequencing technology. However, long-read sequencing is still costly, while short-read sequencing carries less information for complex microbial community profiling; therefore, the applications of high throughput sequencing platforms still remain challenging in microbial community reconstruction analysis. RESULTS: Here, we developed a method to investigate the profile of aligned 16S rRNA gene sequences and to measure the proper region for microbial community reconstruction, as a step in creating a more efficient way to detect microorganism at the genus level. Finally, we found that each genus has its own preferential genus-specific amplicons for a genus assignment, which are not always located in hyper variable regions (HVRs). It was also noted that the rare genera should contribute less than dominant ones to the common profile of the aligned 16S rRNA sequences and have lower affinity to the common universal primer. CONCLUSIONS: Therefore, using multiple 16S rRNA regions rather than one "universal" region can significantly improve the ability of microbial community reconstruction. In addition, we found that a short fragment is suitable for most genera identifications, and the proper conserved regions used for primer design are larger than before.

Exposure to soil, house dust and decaying plants increases gut microbial diversity and decreases serum immunoglobulin E levels in BALB/c mice.

To assess the impact of sanitation of a living environment on gut microbiota and development of the immune system, we raised BALB/c mice under three distinct environmental conditions: a specific pathogen-free animal room (SPF), a general animal room (XZ) and a farmhouse (JD). All other variables like diet, age, genetic background, physiological status and original gut microbiota were controlled for in the three groups. Using high-throughput sequencing of the 16S rRNA gene, we found that each mouse group had a specific structure of the gut microbial community. Groups JD and XZ harboured a significantly more diverse and richer gut microbiota than did group SPF. Bacteroidetes were significantly more abundant in groups XZ and JD than in group SPF, whereas Firmicutes showed the inverse pattern. Total serum immunoglobulin E (IgE) levels were significantly lower in groups XZ and JD than in group SPF. There were no significant differences in gut microbiota diversity and serum IgE concentration between groups JD and XZ, but we found higher abundance of dominant genera in the gut microflora of group JD. We conclude that exposure to soil, house dust and decaying plant material enhances gut microbial diversity and innate immunity. Our results seem to provide new evidence supporting the hygiene hypothesis.

miRNA-miRNA interaction implicates for potential mutual regulatory pattern.

BACKGROUND: Natural or endogenous sense/antisense miRNAs, located on sense and antisense strands in the same genomic region, respectively, are detected recently. However, little is known about these miRNA pairs, especially for their distributions in different animal species. We herein present systematic analysis of them in human, mouse and rat miRNAs, and their expression patterns based on deep sequencing datasets. METHODS AND RESULTS: The phenomenon of miRNA-miRNA interaction could be detected in different animal species. The common miRNAs pairs were found across species. These miRNA pairs could form miRNA:miRNA duplex with complete complementary structure, and were prone to be located on specific chromosomes. They might be homologous miRNA genes (especially in human), or clustered in a gene cluster (especially in rat), or simultaneously detected in different genomic regions due to multicopy pre-miRNAs. Remarkably, some miRNA pairs, located in different genomic regions, also showed complementarity as well as endogenous sense/antisense miRNAs. Based on published deep sequencing datasets, one member of miRNA pairs always was abundantly expressed, whereas another was quite rare. Rare common target mRNAs of these miRNA pairs were predicted. CONCLUSIONS: Interaction between miRNAs and significant expression divergence implied complex potential mutual regulatory pattern in the miRNA world. The study would enrich miRNA regulatory network.

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis.

BACKGROUND: The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. RESULTS: Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. CONCLUSIONS: By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand.

Detection of multiple SNPs in numerous samples with polyacrylamide gel-based microarray.

Previously we have developed polyacrylamide gel-based DNA microarray to genotype single nucleotide polymorphism (SNP) in a large number of samples, which has been proved as a simple and robust platform for high-throughput SNP screening. Here we improved this method to detect multiple SNPs by introducing multiplex polymerase chain reaction (multiplex-PCR) and immobilizing the products of multiplex-PCR to fabricate gel-based microarray and applying universal dual-color detectors in hybridization. In this report, five SNPs (rs191296, rs2280073, rs17599165, rs17599416 and rs7660336) of GABRA4 gene were chosen and successfully analyzed with the improved platform. Our experiment demonstrated that 3-dimentional polyacrylamide gel-based microarray of multiplex-PCR products make the platform for multiple SNPs genotyping becoming more labor-saving and time-saving. Appling the universal dual-color fluorescent detectors can reduce the cost over two-thirds for multiple SNPs analysis. It is concluded that the multiplex-PCR combined with the gel-based microarray hybridized with universal dual-color fluorescent detectors is efficient, rapid and simple for the detection of a single nucleotide mismatch, and may be very competitive in the efficiency, fidelity and cost for constructing DNA microarrays.

Haplotype distribution and evolutionary pattern of miR-17 and miR-124 families based on population analysis.

BACKGROUND: MicroRNAs (miRNAs) are small, endogenously expressed non-coding RNAs that regulate mRNAs post-transcriptionally. Previous studies have explored miRNA evolutionary trend, but evolutionary history and pattern in the miRNA world are still not fully clear. In the paper, we intended to analyze miRNA haplotype distribution and evolutionary network by analyzing miRNA sequences of miR-17 and miR-124 families across animal species as special populations. PRINCIPAL FINDINGS: 31 haplotypes were detected in miR-17 family while only 9 haplotypes were defined in miR-124 family. The complex miR-17 family was mainly distributed in vertebrates, but miR-124 was shared by more animal species from Caenorhabditis to Homo and had a wide distribution spectrum. Some haplotypes of the two miRNA families appeared discontinuous distributions across animals. Compared with a simple phylogenetic network in miR-124 family, miR-17 family indicated a complex network with some median vectors that might be lost ancestral or potential miRNA haplotypes. By analyzing different miRNAs across 12 animal species, we found these small RNAs showed different haplotype diversities, haplotype distributions and phylogenetic networks. CONCLUSIONS: Different miRNAs had quite different haplotype distributions and evolutionary patterns. Discontinuous distributions of miRNAs and median vectors in phylogenetic networks implied more members in the miRNA world. miRNA may be an excellent phylogenetic marker to discover its evolutionary history and pattern across the animal kingdom.

Association study between BDNF gene polymorphisms and autism by three-dimensional gel-based microarray.

Single nucleotide polymorphisms (SNPs) are important markers which can be used in association studies searching for susceptible genes of complex diseases. High-throughput methods are needed for SNP genotyping in a large number of samples. In this study, we applied polyacrylamide gel-based microarray combined with dual-color hybridization for association study of four BDNF polymorphisms with autism. All the SNPs in both patients and controls could be analyzed quickly and correctly. Among four SNPs, only C270T polymorphism showed significant differences in the frequency of the allele (chi(2) = 7.809, p = 0.005) and genotype (chi(2) = 7.800, p = 0.020). In the haplotype association analysis, there was significant difference in global haplotype distribution between the groups (chi(2) = 28.19, p = 3.44e-005). We suggest that BDNF has a possible role in the pathogenesis of autism. The study also show that the polyacrylamide gel-based microarray combined with dual-color hybridization is a rapid, simple and high-throughput method for SNPs genotyping, and can be used for association study of susceptible gene with disorders in large samples.

[Study on the genotyping of single nucleotide polymorphisms for a large number of samples by three-dimensional polyacrylamide gel-based microarray method].

OBJECTIVE: To genotype single nucleotide polymorphisms (SNPs) in a large number of samples by applying three-dimensional polyacrylamide gel-based microarray. METHODS: The method relies on copolymerization of acrylamide-modified PCR products with acrylamide monomers and acryl-modified slides to prepare gel-based microarray. Then array is hybridized with a pair of specific probes and the two universal dual-color fluorescent detectors labeled with Cy3 or Cy5 respectively (Tag1 and Tag2). Electrophoresis is used in post-hybridization to remove the nonspecifically bound targets and mismatches. Finally, genotyping is based on the images captured through two-color fluorescent scanning. RESULTS: The 3-D gel-immobilization of nucleic acids has a high immobilization yield and good hybridization efficiency. As universal dual-color fluorescent detectors are used, it is not required that specific probes be labeled for all SNPs, therefore the expense for synthesis can be reduced considerably. Electrophoresis in post-hybridization can enhance the capability for discriminating a single nucleotide mismatch from the perfectly matched sequence and improve the signal-to-noise ratio significantly. CONCLUSION: The gel-based microarray is a rapid, simple and high-throughput method for SNPs genotyping and may be very competitive in the efficiency, fidelity and cost for constructing DNA microarrays, which will hold significant promise for applications in human DNA diagnostics.

Polyacrylamide gel-based microarray: a novel method applied to the association Study between the polymorphisms of BDNF gene and autism.

Single nucleotide polymorphisms (SNPs) are important markers which can be used in the association study for searching for associated genes of complex diseases. High-throughput methods in a large number of samples are needed for SNP genotyping. In this study, we applied polyacrylamide gel-based microarray combined with universal dual-color hybridization for trios-based associative study of BDNF polymorphisms with autism in Chinese. All four SNPs in both patients and their parents could be analyzed quickly and correctly. In single SNP TDT analysis, C270T showed preferential transmission of the T allele compared to the C allele (TDT p < 0.001) in autism. In haplotype TDT analysis, C270T polymorphism also existed in the haplotype combination which showed significant association (TDT p < 0.05). These results suggest a potential association between BDNF and autism in the Chinese population. The study also show that the polyacrylamide gel-based microarray combined with universal dual-color detectors is a rapid, simple, high-throughput method for SNPs genotyping, and can be very effective and cost effective in association study of susceptible gene with disorders in large samples.