Polymorphic edge detection (PED): two efficient methods of polymorphism detection from next-generation sequencing data.

BACKGROUND: Accurate detection of polymorphisms with a next generation sequencer data is an important element of current genetic analysis. However, there is still no detection pipeline that is completely reliable. RESULT: We demonstrate two new detection methods of polymorphisms focusing on the Polymorphic Edge (PED). In the matching between two homologous sequences, the first mismatched base to appear is the SNP, or the edge of the structural variation. The first method is based on k-mers from short reads and detects polymorphic edges with k-mers for which there is no match between target and control, making it possible to detect SNPs by direct comparison of short-reads in two datasets (target and control) without a reference genome sequence. The second method is based on bidirectional alignment to detect polymorphic edges, not only SNPs but also insertions, deletions, inversions and translocations. Using these two methods, we succeed in making a high-quality comparison map between rice cultivars showing good match to the theoretical value of introgression, and in detecting specific large deletions across cultivars. CONCLUSIONS: Using Polymorphic Edge Detection (PED), the k-mer method is able to detect SNPs by direct comparison of short-reads in two datasets without genomic alignment step, and the bidirectional alignment method is able to detect SNPs and structural variations from even single-end short-reads. The PED is an efficient tool to obtain accurate data for both SNPs and structural variations. AVAILABILITY: The PED software is available at: https://github.com/akiomiyao/ped .

Evaluation of the GeneFields® EHEC/SS PCR dipstick DNA chromatography kit for the detection of enteric bacterial pathogens in stool specimens of healthy humans.

We developed a new GeneFields® EHEC/SS PCR dipstick DNA chromatography kit for the simultaneously detection of invA, ipaH, and stx genes in Salmonella enterica (56 strains), Shigella spp. (44), and enterohemorrhagic Escherichia coli (EHEC) (28), respectively, and evaluated the sensitivity and specificity with other bacteria (57) by this kit. The sensitivity and specificity were 100%, respectively. The detection limit of various methods was determined using 5% (w/v) stool suspensions spiked with each bacterium. The detection limit of the GeneFields® EHEC/SS kit ranged from approximately 102-103 CFU/g. Additionally, the relative sensitivities and specificities of the GeneFields® EHEC/SS kit vs two commercially available real-time PCR kits were >85.0% and >90.0%, respectively. These results indicate that the GeneFields® EHEC/SS kit can be used for genetic screening of S. enterica, Shigella spp., and EHEC in human stool specimens with sensitivities and specificities similar to those of the commercially available real-time PCR kits.

Global transcriptome analysis of pig induced pluripotent stem cells derived from six and four reprogramming factors.

Pigs are important, both for agriculture and as animal models for human diseases. However, due to the lack of embryonic stem cells, the possibility of genetic modification is quite limited. To overcome this limitation, induced pluripotent stem (iPS) cells have been derived from pigs. Despite the public availability of a large number of expression datasets from mice, rats, and primates-derived iPS cells, the expression profile of pig-derived iPS cells is quite limited. Furthermore, there is no dataset focused on the profiling of pig-derived iPS cell with six reprogramming factors (Oct3/4, Sox2, Klf4, c-Myc, Lin28, and Nanog). Here, we used Illumina RNA sequencing platform to characterize the mRNA expression of four-factor derived and six-factor derived pig iPS cells. We observed that the expression levels of whole genes in our established six factors derived iPS cells and parent fibroblast, and compared with that of iPS cells with four factors in public database. These data are valuable in understanding species difference in the reprogramming process of stem cells, and could help identify the key regulating genes involved in the process.

Antisense RNA transcripts in the blood may be novel diagnostic markers for colorectal cancer.

Numerous genetic studies have been conducted regarding the occurrence of colorectal cancer (CRC) and the prognosis using microarrays. However, adequate investigations into the diagnostic application of microarrays have yet to be performed. The simplicity and accuracy of diagnosis and prognosis tracking are important requirements for its processes, and the use of blood cells for diagnosis is considered to be suitable to meet these requirements. The patients involved in the study were 28 preoperative patients with CRC and 6 healthy individuals who served as controls. RNA was extracted from the blood cells of the patients and analyzed using a sense/antisense RNA custom microarray. In the patients with CRC, the expression levels of 20 sense RNA and 20 antisense RNA species were identified as being significantly altered compared with that of the healthy volunteers (P<0.05; fold-change, >2.0). Cluster analysis of these RNA species revealed that the top 10 antisense RNAs significantly clustered patients with cancer and healthy individuals separately. Patients with stage I or II CRC exhibited significant changes in the expression levels of 33 sense and 39 antisense RNA species, as compared with healthy volunteers (P<0.01; fold-change >2.0). Cluster analysis demonstrated that patients with stage I or II CRC and healthy volunteers formed separate clusters only among the top 20 antisense RNA species. A tracking study of expression levels of haloacid dehalogenase-like hydrolase domain-containing 1 (HDHD1) antisense RNA was performed and a significant difference was identified between the CRC and healthy groups revealing that the levels at one week and three months following surgical removal of the cancerous tissue, decreased to almost same levels of the healthy individuals. The results of the current study indicate that HDHD1 antisense RNA may serve as a potential biomarker for the prognosis of CRC.

Genetic structure and relationships of 16 Asian and European cattle populations using DigiTag2 assay.

In this study, we genotyped 117 autosomal single nucleotide polymorphisms using a DigiTag2 assay to assess the genetic diversity, structure and relationships of 16 Eurasian cattle populations, including nine cattle breeds and seven native cattle. Phylogenetic and principal component analyses showed that Bos taurus and Bos indicus populations were clearly distinguished, whereas Japanese Shorthorn and Japanese Polled clustered with European populations. Furthermore, STRUCTURE analysis demonstrated the distinct separation between Bos taurus and Bos indicus (K=2), and between European and Asian populations (K=3). In addition, Japanese Holstein exhibited an admixture pattern with Asian and European cattle (K=3-5). Mongolian (K=13-16) and Japanese Black (K=14-16) populations exhibited admixture patterns with different ancestries. Bos indicus populations exhibited a uniform genetic structure at K=2-11, thereby suggesting that there are close genetic relationships among Bos indicus populations. However, the Bhutan and Bangladesh populations formed a cluster distinct from the other Bos indicus populations at K=12-16. In conclusion, our study could sufficiently explain the genetic construction of Asian cattle populations, including: (i) the close genetic relationships among Bos indicus populations; (ii) the genetic influences of European breeds on Japanese breeds; (iii) the genetic admixture in Japanese Holstein, Mongolian and Japanese Black cattle; and (iv) the genetic subpopulations in Southeast Asia.

Discovery, primary, and crystal structures and capacitation-related properties of a prostate-derived heparin-binding protein WGA16 from boar sperm.

Mammalian sperm acquire fertility through a functional maturation process called capacitation, where sperm membrane molecules are drastically remodeled. In this study, we found that a wheat germ agglutinin (WGA)-reactive protein on lipid rafts, named WGA16, is removed from the sperm surface on capacitation. WGA16 is a prostate-derived seminal plasma protein that has never been reported and is deposited on the sperm surface in the male reproductive tract. Based on protein and cDNA sequences for purified WGA16, it is a homologue of human zymogen granule protein 16 (ZG16) belonging to the Jacalin-related lectin (JRL) family in crystal and primary structures. A glycan array shows that WGA16 binds heparin through a basic patch containing Lys-53/Lys-73 residues but not the conventional lectin domain of the JRL family. WGA16 is glycosylated, contrary to other ZG16 members, and comparative mass spectrometry clearly shows its unique N-glycosylation profile among seminal plasma proteins. It has exposed GlcNAc and GalNAc residues without additional Gal residues. The GlcNAc/GalNAc residues can work as binding ligands for a sperm surface galactosyltransferase, which actually galactosylates WGA16 in situ in the presence of UDP-Gal. Interestingly, surface removal of WGA16 is experimentally induced by either UDP-Gal or heparin. In the crystal structure, N-glycosylated sites and a potential heparin-binding site face opposite sides. This geography of two functional sites suggest that WGA16 is deposited on the sperm surface through interaction between its N-glycans and the surface galactosyltransferase, whereas its heparin-binding domain may be involved in binding to sulfated glycosaminoglycans in the female tract, enabling removal of WGA16 from the sperm surface.

Mode of swine hepatitis E virus infection and replication in primary human hepatocytes.

The aim of this study was to investigate the infection and replication of swine-derived hepatitis E virus (HEV) in primary cultured human hepatocytes (PHCs). Hepatocytes were cultured from the resected normal livers of patients with metastatic tumours. These cultured hepatocytes were infected with swine-derived genotype 3 or 4 HEV. Viral replication was monitored using reverse transcriptase-quantitative PCR. The amount of HEV RNA increased in the culture media and cells following infection. Immunofluorescence staining implied that the spread of HEV infection in hepatocytes was attributed mainly to cell-to-cell transmission via the cell membrane. The sequences of the inoculated and propagated HEV were determined to examine whether sequence variation occurred during infection. Sequence analysis showed that there were no differences between inoculated and propagated HEV, demonstrating that in vitro infection and replication of swine HEV in PHCs occurred without sequence variation.

Tag/hybridization-based sensitive detection of polymerase chain reaction products.

The polymerase chain reaction (PCR) is an important technology to amplify a single copy or a few copies of DNA segment in genomic DNAs, visualizing the segment as DNA fragment. Thus, PCR is frequently used in various examinations such as detection of bacteria and fungi in the food industry. Here, we report a simple and sensitive method for detection of PCR products using single-strand tag sequence and hybridization of the tag sequence to the complementary tag sequence immobilized on solid material (STH). The detection sensitivity was found to be at least 50 times higher than electrophoresis/ethidium bromide (EtBr) visualization for approximately a 500-bp fragment and higher than the ordinary hybridization, that is, hybridization of denatured PCR product to probe sequence immobilized on solid material.

Transposon Insertion Finder (TIF): a novel program for detection of de novo transpositions of transposable elements.

BACKGROUND: Transposition event detection of transposable element (TE) in the genome using short reads from the next-generation sequence (NGS) was difficult, because the nucleotide sequence of TE itself is repetitive, making it difficult to identify locations of its insertions by alignment programs for NGS. We have developed a program with a new algorithm to detect the transpositions from NGS data. RESULTS: In the process of tool development, we used next-generation sequence (NGS) data of derivative lines (ttm2 and ttm5) of japonica rice cv. Nipponbare, regenerated through cell culture. The new program, called a transposon insertion finder (TIF), was applied to detect the de novo transpositions of Tos17 in the regenerated lines. TIF searched 300 million reads of a line within 20 min, identifying 4 and 12 de novo transposition in ttm2 and ttm5 lines, respectively. All of the transpositions were confirmed by PCR/electrophoresis and sequencing. Using the program, we also detected new transposon insertions of P-element from NGS data of Drosophila melanogaster. CONCLUSION: TIF operates to find the transposition of any elements provided that target site duplications (TSDs) are generated by their transpositions.

The synthesis of cytosolic ascorbate peroxidases in germinating seeds and seedlings of soybean and their behavior under flooding stress.

Cytosolic ascorbate peroxidases (cAPXs) of soybean have been found by proteome analysis to be downregulated in submerged seedlings. To elucidate the physiological meaning of this downregulation, soybean cAPXs were characterized in this study. Vigorous synthesis was detected in germinating seeds and seedlings. Expression of the corresponding genes was detected clearly in tissues that actively underwent cell division. The gene expression was suppressed by flooding stress, but not by salinity, cold or drought stress. The expression recovered 1 d after release from flooding stress, accompanied by growth resurgence.

Proteomic and biochemical analyses of the cotyledon and root of flooding-stressed soybean plants.

BACKGROUND: Flooding significantly reduces the growth and grain yield of soybean plants. Proteomic and biochemical techniques were used to determine whether the function of cotyledon and root is altered in soybean under flooding stress. RESULTS: Two-day-old soybean plants were flooded for 2 days, after which the proteins from root and cotyledon were extracted for proteomic analysis. In response to flooding stress, the abundance of 73 and 28 proteins was significantly altered in the root and cotyledon, respectively. The accumulation of only one protein, 70 kDa heat shock protein (HSP70) (Glyma17g08020.1), increased in both organs following flooding. The ratio of protein abundance of HSP70 and biophoton emission in the cotyledon was higher than those detected in the root under flooding stress. Computed tomography and elemental analyses revealed that flooding stress decreases the number of calcium oxalate crystal the cotyledon, indicating calcium ion was elevated in the cotyledon under flooding stress. CONCLUSION: These results suggest that calcium might play one role through HSP70 in the cotyledon under flooding stress.

Differential expression of PRAMEL1, a cancer/testis antigen, during spermatogenesis in the mouse.

PRAME belongs to a group of cancer/testis antigens (CTAs) that are characterized by their restricted expression in normal gametogenic tissues and a variety of tumors. The PRAME family is one of the most amplified gene families in the mouse and other mammalian genomes. Members of the PRAME gene family encode leucine-rich repeat (LRR) proteins functioning as transcription regulators in cancer cells. However, the role of PRAME in normal gonads is unknown. The objective of this study is to characterize the temporal and spatial expression of the mouse Pramel1 gene, and to determine the cellular localization of the PRAMEL1 protein during the mouse spermatogenesis. Our results indicated that the mouse Pramel1 was expressed in testis only. The mRNA and protein expression level was low in the newborn testes, and gradually increased from 1- to 3-week-old testes, and then remained constant after three weeks of age. Immunofluorescent staining on testis sections with the mouse PRAMEL1 antibody revealed that PRAMEL1 was localized in the cytoplasm of spermatocytes and the acrosomal region of round, elongating and elongated spermatids. Further analyses on the testis squash preparation and spermatozoa at a subcellular level indicated that the protein localization patterns of PRAMEL1 were coordinated with morphological alterations during acrosome formation in spermatids, and were significantly different in connecting piece, middle piece and principal piece of the flagellum between testicular and epididymal spermatozoa. Collectively, our results suggest that PRAMEL1 may play a role in acrosome biogenesis and sperm motility.

Examination of the mouse embryo by micro-CT.

Micro X-ray computed tomography (micro-CT) is widely used in preclinical studies of small animals. However, due to the low soft tissue contrast, segmentation of soft tissues in the micro-CT image is a challenging problem. To gain a better understanding of the macroscopic anatomy of the mouse embryo, 3 fixation methods and 3 metal stainings were examined for micro-CT using C57BL/6J mouse embryos in the present study. The examination demonstrated that 1% acetic acid/95% ethanol fixative together with zinc staining provided a high contrast micro-CT image, enabling the segmentation of soft tissues. Then, using this condition, the macroscopic embryo structure of the nude mouse was examined, revealing lack of a thymus. It appears that micro-CT with the fixation and staining condition devised in the present study could be a powerful tool in detecting the effects of various mutations at embryonic stages.

Gene expression profiling of sense and antisense transcripts in liver regeneration by microarray analysis.

Liver regeneration is a hyperplastic phenomenon induced by partial hepatectomy (PH) or hepatic damage. A large number of genes have been indicated to be involved in the process of liver regeneration. It was recently reported that natural antisense transcripts are involved in the regulation of gene expression. However, no antisense transcript expressions in liver regeneration have been reported thus far. Therefore, the present study aimed to comprehensively identify up- or downregulated sense and antisense transcripts in liver regeneration using PH mice and a sense/antisense custom-microarray. The results showed that 97 genes were upregulated and 7 genes were downregulated for sense transcripts, whereas 15 genes were upregulated and 2 genes were downregulated for antisense transcripts in regenerating livers as compared to normal livers (P<0.05 and fold change >2.0). Sense and antisense transcripts of the genes, Apoa4, Hp, Fgb and Fgg, exhibited concordant upregulation during the course of liver regeneration. Apoa4, Hp and Fgb transcripts were further investigated by strand-specific reverse transcription-quantitative polymerase chain reaction (RT-qPCR), revealing results consistent with those of the microarray. In conclusion, the up- or downregulated sense and antisense transcripts identified in the present study are suggested to be involved in liver regeneration.

Pig sperm membrane microdomains contain a highly glycosylated 15-25-kDa wheat germ agglutinin-binding protein.

A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both N- and O-glycans consisting of neutral monosaccharides. Interestingly, WGA-gp may be involved in intracellular Ca(2+) regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca(2+) oscillation without changing the basal intracellular Ca(2+) concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca(2+) concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca(2+) regulation may be a common feature among animal sperm.

Differential expression profiles of sense and antisense transcripts between HCV-associated hepatocellular carcinoma and corresponding non-cancerous liver tissue.

Recent studies have demonstrated that natural antisense transcripts, which are complementary sequences to messenger RNA, have important cellular functions such as the stabilization and silencing of mRNA. However, the possible contribution of antisense transcripts in hepatocellular carcinoma (HCC) development has not been described. Therefore, we simultaneously investigated the sense and antisense transcripts of HCC and non-cancerous tissues to explore the possible contribution of antisense transcripts to HCC progression. RNA was prepared from 15 HCV-associated HCCs and from 6 corresponding non-cancerous tissues and was subjected to expression profile analysis of sense and antisense transcripts using a human custom microarray. Differential expression of 161 sense and 25 antisense transcripts was observed with more than 2-fold between HCC and non-cancerous tissue (p<0.001). The expression of the sense and antisense transcripts was used to cluster cancer and non-cancerous tissues, and the cancer and non-cancerous tissues were found to be clearly separated into different clusters. Additionally, the sense and antisense expression profiles were analyzed with regard to HCC differentiation (p<0.001), resulting in 71 sense and 43 antisense transcripts. These unique transcripts did not overlap with those found in the discrimination of HCC from non-cancerous tissues. When the HCC tissues were clustered by transcript expression, the antisense transcripts resulted in clustering of HCC that was consistent with grouping based on histology. These findings strongly indicate that the antisense transcripts together with the sense transcripts are involved in liver tumorigenesis.

Identification of up-regulated and down-regulated cis-natural antisense transcripts in the human B lymphoblastic cell line IM-9 after X-ray irradiation.

Ionizing radiation (IR) causes DNA injury and induces multiple signal mechanisms, including the regulation of DNA repair, the cell cycle and gene expression through the activation of p53-related pathways. Cis-natural antisense transcripts (cis-NATs), which are transcribed from the DNA strand opposite to that for mRNA of the gene, are recognized as important regulators of gene expression in eukaryotic cells, but the effects on cis-NAT expression by IR are unknown to date. Therefore, we investigated the effects of X-ray irradiation on cis-NAT expression together with mRNA expression using a human B lymphoblast cell line (IM-9), a custom-microarray and strand-specific RT-qPCR. Eighteen, 33 and 106 mRNAs were demonstrated to be differentially expressed in IM-9 cells after 1, 2 and 4 Gy irradiation, respectively, as compared to 0 Gy by microarray analysis (fold change, FC >2.0). On the other hand, 10, 22 and 43 NATs were demonstrated to be differentially expressed in IM-9 cells after 1, 2 and 4 Gy irradiation, respectively, as compared to 0 Gy by microarray analysis (FC >2.0). Among these mRNAs/NATs, the IR dose-dependent up-regulation of mRNAs and cis-NATs of MDM2 and CDKN1A were confirmed by strand-specific RT-qPCR. Additionally, the cis-NATs of MDM2 were indicated to be localized in the cytoplasm, while cis-NATs of CDKN1A were located in the nucleus and cytoplasm. In conclusion, the radiation-responsive cis-NATs in conjunction with mRNAs were identified for the first time in the present study. It is possible that these cis-NATs regulate the gene expression in a post-transcriptional fashion. The IR dose-dependent up- and down-regulation of these mRNAs/cis-NATs may be a marker for ionizing radiation.

Molecular cloning of the swine interleukin-23 subunit p19 and of its receptor components interleukin-23Rα and -12Rß1.

Interleukin (IL)-12 and IL-23 play central roles in the regulation of distinct helper T-cell subsets, i.e. Th1 and Th17, respectively. Although IL-12 and IL-23 have been well studied in human and rodent systems, little is known about their significance in other animals, including livestock mammals such as cattle and pigs. In this study, we performed molecular cloning and genetic characterization of a small component of swine IL-23, i.e., IL-23p19; in addition, we identified and performed chromosomal assignment of the genes encoding its receptor (R) subunits IL-23Rα and IL-12Rß1. These results provide genetic information about both swine IL-23/IL-23R and IL-12/IL-12R systems, which allows for better understanding of IL-12/IL-23 systems involved in pig immunity.

Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots.

Alcohol dehydrogenase (Adh) is the key enzyme in alcohol fermentation. We analyzed Adh expression in order to clarify the role of Adh of soybeans (Glycine max) to flooding stress. Proteome analysis confirmed that expression of Adh is significantly upregulated in 4-day-old soybean seedlings subjected to 2 days of flooding. Southern hybridization analysis and soybean genome database search revealed that soybean has at least 6 Adh genes. The GmAdh2 gene that responded to flooding was isolated from soybean cultivar Enrei. Adh2 expression was markedly increased 6 h after flooding and decreased 24 h after floodwater drainage. In situ hybridization and Western blot indicated that flooding strongly induces Adh2 expression in RNA and protein levels in the root apical meristem. Osmotic, cold, or drought stress did not induce expression of Adh2. These results indicate that Adh2 is a flooding-response specific soybean gene expressed in root tissue.

Transcriptional responses to flooding stress in roots including hypocotyl of soybean seedlings.

To understand the transcriptional responses to flooding stress in roots including hypocotyl of soybean seedlings, genome-wide changes in gene expression were analyzed using a soybean microarray chip containing 42,034 60-mer oligonucleotide probes. More than 6,000 of flooding-responsive genes in the roots including hypocotyl of soybean seedlings were identified. The transcriptional analysis showed that genes related to photosynthesis, glycolysis, Ser-Gly-Cys group amino acid synthesis, regulation of transcription, ubiquitin-mediated protein degradation and cell death were significantly up-regulated by flooding. Meanwhile, genes related to cell wall synthesis, secondary metabolism, metabolite transport, cell organization, chromatin structure synthesis, and degradation of aspartate family amino acid were significantly down-regulated. Comparison of the responses with other plants showed that genes encoding pyrophosphate dependent phosphofructokinase were down-regulated in flooded soybean seedlings, however, those in tolerant plants were up-regulated. Additionally, genes related to RNA processing and initiation of protein synthesis were not up-regulated in soybean, however, those in tolerant plants were up-regulated. Furthermore, we found that flooding-specific up-regulation of genes encoding small proteins which might have roles in acclimation to flooding. These results suggest that functional disorder of acclimative responses to flooding through transcriptional and post-transcriptional regulations is involved in occurring flooding injury to soybean seedlings.