Solexa Sequencing Research Articles

Analysis of Serum microRNA Profile by Solexa Sequencing in Women With Endometriosis

The potential roles of serum microRNAs (miRNAs), as biomarkers, in noninvasive diagnosis of endometriosis have been reported by microarray analysis. However, microarray analysis cannot perform well in outcome accuracy and repeatability and is not suitable to be used for exploring new targets. Here, Solexa sequencing, a wide and precise method, was adopted to further analyze the serum miRNAs profile in endometriosis, which may offer more evidence to apply serum miRNAs as biomarkers in diagnosis of endometriosis. Serum samples were collected from 30 patients with minimal-mild endometriosis and 20 women without endometriosis as control. Expression of serum miRNAs was measured by Solexa sequencing and validated by quantitative real-time polymerase chain reaction (qPCR). Solexa sequencing showed 93.63% clean readouts for all small RNAs in the serum of patients with endometriosis and controls. A total of 108 miRNAs were found to be differentially expressed in the serum of patients with endometriosis by deep sequencing, compared to controls. Among them, 98 miRNAs were significantly downregulated, while 10 miRNAs were significantly upregulated. Only 21 of 98 significantly downregulated miRNAs, and none of significantly upregulated miRNAs were reported in published literatures, which may be due to the differences in samples and analytical methods. The Solexa sequencing results were consequently validated by qPCR in additional samples. Some miRNAs were identified to be promising diagnostic markers of endometriosis. The functional annotation of target genes revealed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that a majority of differential miRNAs might be involved in endometriosis. Circulating miRNAs may be useful as detection biomarkers for the early diagnosis of minimal-mild endometriosis.

Discovery of putative capsaicin biosynthetic genes by RNA-Seq and digital gene expression analysis of pepper.

The Indian pepper ‘Guijiangwang’ (Capsicum frutescens L.), one of the world’s hottest chili peppers, is rich in capsaicinoids. The accumulation of the alkaloid capsaicin and its analogs in the epidermal cells of the placenta contribute to the pungency of Capsicum fruits. To identify putative genes involved in capsaicin biosynthesis, RNA-Seq was used to analyze the pepper’s expression profiles over five developmental stages. Five cDNA libraries were constructed from the total RNA of placental tissue and sequenced using an Illumina HiSeq 2000. More than 19 million clean reads were obtained from each library, and greater than 50% of the reads were assignable to reference genes. Digital gene expression (DGE) profile analysis using Solexa sequencing was performed at five fruit developmental stages and resulted in the identification of 135 genes of known function; their expression patterns were compared to the capsaicin accumulation pattern. Ten genes of known function were identified as most likely to be involved in regulating capsaicin synthesis. Additionally, 20 new candidate genes were identified related to capsaicin synthesis. We use a combination of RNA-Seq and DGE analyses to contribute to the understanding of the biosynthetic regulatory mechanism(s) of secondary metabolites in a nonmodel plant and to identify candidate enzyme-encoding genes.

Comparative profiling of differentially expressed microRNAs between the follicular and luteal phases ovaries of goats

To explore if the regulation at post-transcriptional level of follicular phase (Fols) to luteal phase (Luts) transition occurs in the ovaries of Anhuai goats, the differentially expressed microRNAs (miRNAs) of ovaries in the Fols and Luts were analyzed using Solexa sequencing in the study. In total, 320 known miRNAs were co-expressed in the two phases, 339 and 353 known miRNAs were expressed in the ovary in the Fols and Luts, respectively. In addition, 45 novel miRNAs were co-expressed in the two phases, 70 and 94 novel miRNAs were expressed in the ovary in the Fols and Luts, respectively. Let-7f was the highest expressed significantly different known miRNA in the two phases, and mir-159 was the highest expressed significantly different novel miRNA in the two phases, which may participate in the follicular-luteal transition of Anhuai goats. GO annotation and KEGG pathway analysis were applied to analyze the target genes of differentially expressed miRNAs detected in the two phases. The results will help to further understand the role of miRNAs in the regulation of follicular to luteal transition in goat ovaries.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2902-1) contains supplementary material, which is available to authorized users.

Identification and differential expression of microRNAs in the ovaries of pigs (Sus scrofa) with high and low litter sizes.

Litter size affects profitability in the swine industry. Mammalian ovaries play important roles during reproduction, including ovulation and hormone secretion, which are tightly regulated by specific microRNAs (miRNAs). In this study, we investigated the effects of specific miRNAs on porcine litter size. We compared the ovarian miRNAs of Yorkshire pigs with high (YH) and low (YL) litter sizes using Solexa sequencing technology. We identified 327 and 320 miRNAs in the ovaries of YH and YL pigs respectively. A total of 297 miRNAs were co-expressed; 30 and 23 miRNAs respectively were specifically expressed in the two libraries. A total of 83 novel miRNAs were predicted; 37 specific miRNAs were obtained, of which 21 miRNAs were upregulated and 16 miRNAs were downregulated in YH compared with YL. Additionally, 19628 and 19250 target genes were predicted in the two libraries respectively. The results revealed that specific miRNAs (i.e., miR-224, miR-99a, let-7c, miR-181c, miR-214 and miR-21) may affect porcine litter size. The results of this study will help in gaining understanding of the role of miRNAs in porcine litter size regulation.

Transcriptome profiling and characterization of gene families with zinc finger and nucleotide binding site (NBS) domains in Larix kaempferi

Larix kaempferi is widely cultivated because of its significant ecological and economic values. However, the molecular mechanism underlying the growth and development of Larix kaempferi remains largely unknown because of its long growth cycle and highly complex genetic background. In this study, global transcriptome sequencing of Larix kaempferi was conducted via Solexa sequencing. In total, 15.49 million clean reads were assembled into 20,395 unigenes and then annotated. The study found hundreds of highly expressed unigenes involved in growth and development (e.g. plant hormone regulation pathways or starch and sucrose metabolic pathways), of which 25 unigenes with full-length coding regions interested us the most. In addition, two gene families containing the zinc finger and nucleotide-binding site (NBS) domains were further explored. A total of 48 C3H-type zinc finger genes, most containing a conversed C-X7–10-C-X5-C-X3-H motif, were identified and divided into eight distinct subfamilies through phylogenetic analysis. Twenty-one genes containing the NBS domain represented four subfamilies. These findings provided new insights into the transcriptome characteristics of Larix kaempferi and abundant sequence information to elucidate the roles of the functional genes in Larix kaempferi.

Changes in the Expression of Serum MiR-887-5p in Patients With Endometrial Cancer.

The purpose of the study was to examine whether miR-887-5p could be used as a diagnostic marker for endometrial cancer. In the first stage, differentially expressed serum micro-RNAs (miRNAs) in the sera of 50 healthy subjects and 50 patients with endometrial cancer were screened by performing Solexa sequencing. In addition, differential expression of these serum miRNAs in endometrial cancer tissues and adjacent normal tissues from 3 patients with endometrial cancer was examined. Comparison of the differentially expressed miRNAs showed that miR-887-5p was significantly expressed in the sera of patients with endometrial cancer as well as in endometrial cancer tissues. In the second stage, quantitative reverse transcription polymerase chain reaction was performed to verify the levels of miR-887-5p in the sera of 20 patients with endometrial cancer and of 20 healthy subjects. Expression of miR-887-5p was significantly increased in the sera of patients with endometrial cancer (P < 0.05) compared with that in the sera of healthy subjects. Receiver operating characteristic curve analysis showed that the area of miR-887-5 under the ROC curve for endometrial cancer diagnosis was 0.728, specificity was 0.60, sensitivity was 0.95, and 95% confidence interval was 0.563-0.892. Besides, with strict screen, we eliminate the other risk factors of endometrial cancer in our healthy donors and cancer patients. Statistical analysis of data obtained for patients in the 2 stages by using SPSS version 17.0 indicated that menarche age (P = 0.47, P = 0.49), body mass index (P = 0.313, P = 0.749), history of hypertension (P = 0.517, P = 0.058), and diabetes (P = 0.205, P = 0.507) had no correlation with endometrial cancer. Serum miR-887-5p levels were significantly increased in patients with endometrial cancer. Therefore, serum miR-887-5p could be used as a potential biomarker for endometrial cancer.

Dysregulated miR-133a Mediates Loss of Type II Collagen by Directly Targeting Matrix Metalloproteinase 9 (MMP9) in Human Intervertebral Disc Degeneration.

A microRNA (miRNA) study using Solexa sequencing. The purpose of this study was to identify intervertebral disc degeneration (IDD)-specific miRNA expression profile, and to validate its biological function. Accumulating evidence indicates that miRNAs play a critical role in IDD, but the role of specific miRNAs involved in this entity remains unclear. MiRNA expression profile was determined in nucleus pulposus (NP) tissues from patients with IDD and controls, employing Solexa sequencing and quantitative real-time PCR (qRT-PCR). Biological functions of differential expression miRNAs were further investigated. Luciferase reporter assays and western blotting were performed to determine miRNA targets. We identified 31 miRNAs that were differentially expressed (22 upregulated and nine downregulated) in patients compared with controls. After qRT-PCR confirmation, miR-133a was significantly down-regulated in degenerative NP tissues. Moreover, its level was inversely correlated with grade of disc degeneration. Through gain- and loss-of-function studies, miR-133a was demonstrated to significantly promote type II collagen expression in NP cells. MMP9 was identified as a target of miR-133a. Knockdown of MMP9 induced effects on NP cells similar to those induced by miR-133a. Expression of MMP9 was inversely correlated with miR-133a expression in degenerative NP tissues. These results suggest that the downregulation of miR-133a induces type II collagen loss by directly targeting MMP9. Our findings also highlight miR-133a as a novel hopeful therapeutic target for IDD. 3.

Genome‐wide identification and characterization of Eutrema salsugineummicroRNAs for salt tolerance

Eutrema salsugineum, a close relative of Arabidopsis thaliana, is a valuable halophytic model plant that has extreme tolerance to salinity. As posttranscriptional gene regulators, microRNAs (miRNAs) control gene expression and a variety of biological processes, including plant-stress responses. To identify salt-stress responsive miRNAs in E. salsugineum and reveal their possible roles in the adaptive response to salt stress, we chose the Solexa sequencing platform to screen the miRNAs in 4-week-old E. salsugineum seedlings under salt treatment. A total of 82 conserved miRNAs belonging to 27 miRNA families and 17 novel miRNAs were identified and 11 conserved miRNA families and 4 novel miRNAs showed a significant response to salt stress. To investigate the possible biological roles of miRNAs, 1060 potential targets were predicted. Moreover, 35 gene ontology (GO) categories and 1 pathway, including a few terms that were directly and indirectly related to salt stress, were significantly enriched in the salt-stress-responsive miRNAs targets. The relative expression analysis of six target genes was analyzed using quantitative real-time polymerase chain reaction (PCR) and showed a negative correlation with their corresponding miRNAs. Many stress regulatory and phytohormone regulatory cis-regulatory elements were widely present in the promoter region of the salt-responsive miRNA precursors. This study describes the large-scale characterization of E. salsugineum miRNAs and provides a useful resource for further understanding of miRNA functions in the regulation of the E. salsugineum salt-stress response.

The chemical toxicity of cesium in Indian mustard (Brassica juncea L.) seedlings

To distinguish between the radiological and chemical effects of radiocesium, we study the chemical toxicity of cesium in the seedlings of Indian mustard (Brassica juncea L.). In this study, the experiment was designed in two factors and five levels random block design to investigate the interaction effects of Cs and K. Results showed that excessive Cs was one of the main factors influence the growth of Brassica juncea seedlings. And the toxicity of Cs in Brassica juncea is likely to be caused by Cs interacts with K-binding sites in essential K-dependent protein, either competes with K for essential biochemical functions, causing intracellular metabolic disturbance. To test the hypothesis that the toxicity of Cs might cause intracellular metabolic disturbance, next-generation sequencing (NGS)-based Illumina paired-end Solexa sequencing platform was employed to analysis the changes in gene expression, and understand the key genes in B. juncea seedlings responding to the toxicity of Cs. Based on the assembled de novo transcriptome, 2032 DEGs that play significant roles in the response to the toxicity of Cs were identified. Further analysis showed that excessive Cs is disturbance the auxin signal transduction pathway, and inhibited the indoleacetic acid-induced protein (AUX/IAA) genes expression eventually lead the seedlings growth and development be inhibited. The results suggest that disturbances to tryptophan metabolism might be linked to changes in growth.

Identification of Serum miR-146a and miR-155 as Novel Noninvasive Complementary Biomarkers for Ankylosing Spondylitis.

Prospective serum microRNAs study. To investigate differentially expressed serum microRNAs (miRNAs) between ankylosing spondylitis (AS) patients and controls, and to evaluate the potential of miRNAs as biomarkers for AS. There is an urgent need to explore novel biomarkers with more high sensitivity and specificity for the diagnosis of AS, especially for early-stage AS. Recently, the discovery of miRNAs has paved a new avenue for the diagnosis of cancers and other diseases, However, the global serum miRNAs pattern in AS patients has never been determined. An initial screening of miRNA expression by Solexa sequencing was performed using serum samples pooled from 10 AS patients and 10 controls, respectively. Subsequently, differential expression was validated using hydrolysis probe-based stem-loop quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, AS patients were divided into group A (kyphosis <70°) and group B (kyphosis ≥70°). Disease activity and functional status were evaluated by Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and the Bath Ankylosing Spondylitis Functional Index (BASFI), respectively. The areas under the receiver-operating characteristic (ROC) curves of identified miRNAs were analyzed. Nineteen serum miRNAs were differentially expressed in AS patients compared with controls. After qRT-PCR confirmation, miR-146a and miR-155 were significantly upregulated in AS patients. The areas under the ROC curves using miR-146a and miR-155 were 0.917 and 0.964, respectively. Importantly, the miR-155 expression level in group B was significantly higher than that in group A. In addition, significant correlation was observed between miR-155 expression level and BASDAI (r = 0.5, P < 0.01). Detection of serum miRNAs (miR-146a and miR-155) may serve as novel complementary biomarkers for AS. Moreover, the expression level of miR-155 is suggested to be associated with disease activity and the severity of thoracolumbar kyphosis secondary to AS. NA.

MiR-sc3, a Novel MicroRNA, Promotes Schwann Cell Proliferation and Migration by Targeting Astn1.

MicroRNAs (miRNAs, miRs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level. We have previously identified a group of novel miRNAs in proximal sciatic nerve after sciatic nerve transection by Solexa sequencing, and miR-sc3 is a member of the group. In this study, we aimed to investigate the effects of miR-sc3 on phenotypic modulation of Schwann cells (SCs). miR-sc3 was highly expressed in the injured nerve after sciatic nerve transection. An increased and decreased expression of miR-sc3 promoted and reduced the proliferation and migration of primary SCs, respectively. miR-sc3 directly targeted astrotactin 1 (Astn1) and led to translational suppression of Astn1. There was an inverse association between the time-dependent expressions of miR-sc3 and Astn1 in proximal sciatic nerve after sciatic nerve transection. Overall, miR-sc3 affected SC proliferation and migration by targeting Astn1, thus playing the regulatory role in peripheral nerve regeneration.

A group of novel serum diagnostic biomarkers for multidrug‐resistant tuberculosis by iTRAQ‐2D LC‐MS/MS and Solexa sequencing

The epidemic of pulmonary tuberculosis (TB), especially multidrug‐resistance tuberculosis (MDR‐TB) presented a major challenge for TB treatment today. We performed iTRAQ labeling coupled with two‐dimensional liquid chromatography‐tandem mass spectrometry (2D LC‐MS/MS) and Solexa sequencing among MDR‐TB patients, drug‐sensitive tuberculosis (DS‐TB) patients, and healthy controls. A total of 50 differentially expressed proteins and 43 differentially expressed miRNAs (fold change &gt;1.50 or &lt;0.60, P&lt;0.05) were identified in the MDR‐TB patients compared to both DS‐TB patients and healthy controls. We found that 22.00% of differentially expressed proteins and 32.56% of differentially expressed miRNAs were related, and could construct a network mainly in complement and coagulation cascades. Significant differences in CD44 antigen (CD44), coagulation factor XI (F11), kininogen‐1 (KNG1), miR‐4433b‐5p, miR‐424‐5p, and miR‐199b‐5p were found among MDR‐TB patients, DS‐TB patients and healthy controls (P&lt;0.05) by enzyme‐linked immunosorbent assay (ELISA) and SYBR green qRT‐PCR validation. A strong negative correlation, consistent with the target gene prediction, was found between KNG1 and miR‐199b‐5p (r=−0.232, P=0.017). Moreover, we established a diagnostic model for MDR‐TB patients and healthy controls with a sensitivity of 100.00% and a specificity of 88.33% (overall accuracy 93.14%), and also established a diagnostic model for MDR‐TB patients and DS‐TB patients with a sensitivity of 83.33% and a specificity of 88.33% (overall accuracy 86.27%). Our study proposes potential biomarkers for MDR‐TB diagnosis, and also provides a new experimental basis to understand the pathogenesis of MDR‐TB.Support or Funding InformationThis work was supported by grants from the National Natural Science Foundation of China (No.81573709, No.81273882), the National Basic Research Program of China (No.2014CB543002) and the National Special Sci‐Tech Projects (No.2012ZX10005001‐006).

Identification of miRNAs in sweet potato by Solexa sequencing

MicroRNAs (miRNAs) are small endogenous RNAs, involved in plant growth and development as well as in stress responses. Among them, some are highly evolutionally conserved in the plant kingdom, which provides a powerful strategy for identifying miRNAs in a new species. Sweet potato (Ipomoea batatas L.) is one of the important food crops in the world, but few of its miRNAs have been determined. In this study, a total of 24 conserved miRNAs belonged to 14 miRNA families, and 16 novel miRNAs were identified by deep sequencing. Using previously established protocols, a total of 48 potential target genes were predicted for the conserved and novel miRNAs. These target genes are involved in transcription, metabolism, defense response, oxidationreduction processes, etc. Overall, this study provides information concerning the miRNAs precursors in I. batatas, mature miRNAs, and miRNA targets, and it is valuable as the basis for the future research of miRNA functions in I. batatas.

Complete genome sequence and comparative genome analysis of the Paenibacillus mucilaginosus K02

AimPaenibacillus mucilaginosus (P. mucilaginosus) K02 is implicated in mineral weathering. However, relevant molecular mechanisms remain obscure. The study aims to uncover the bacterium's physiological processes using genomic approaches. Methods and resultsGenomic DNA from P. mucilaginosus K02 was sequenced using high-throughput Solexa sequencing technology and then conducted for Clusters of Orthologous Group (COG) annotation. Thereafter, genome sequences of K02 were compared with two strains, 3016 and KNP414. Mummer was applied for collinearity analysis of three P. mucilaginosus genomes. BLAST was used to identify pan and core genes in these strains. Finally, a phylogenetic tree was constructed using the maximum likelihood method by TreeBeST. Complete genome sequence of P. mucilaginosus K02 indicated the strain comprises one circular chromosome with 8,819,200 bases containing 58.3% GC content and 84.75% coding regions. A total of 7299 predicted ORFs were identified in the genome, among them, several genes were related to carbonic anhydrase (CA), and exopolysaccharide biosynthesis and secretion. Moreover, proteins of the predicted genes were annotated in COG categories such as “Carbohydrate transport and metabolism” and “Inorganic ion transport and metabolism.” In comparison with KNP414 and 3016, K02 exhibited chromosomal recombination or transposition. A total of 6662 core genes were identified among three P. mucilaginosus strains. The phylogenomic study indicated that P. mucilaginosus K02 was clustered with P. mucilaginosus strains 3016 and KNP414. ConclusionsIn P. mucilaginosus K02, genes related to CA and exopolysaccharide biosynthesis and secretion, and that involved in metabolism-related processes might play significant roles in mineral weathering.

Identification of Novel and Conserved miRNAs in Leaves of In vitro Grown Citrus reticulata “Lugan” Plantlets by Solexa Sequencing

MicroRNAs (miRNAs) play essential roles in plant development, but the roles in the in vitro plant development are unknown. Leaves of ponkan plantlets derived from mature embryos at in vitro culture conditions were used to sequence small RNA fraction via Solexa sequencing, and the miRNAs expression was analyzed. The results showed that there were 3,065,625 unique sequences in ponkan, of which 0.79% were miRNAs. The RNA sequences with lengths of 18–25 nt derived from the library were analyzed, leading to the identification of 224 known miRNAs, of which the most abundant were miR157, miR156, and miR166. Three hundred and fifty-eight novel miRNA candidates were also identified, and the number of reads of ponkan novel miRNAs varied from 5 to 168,273. The expression of the most known miRNAs obtained was at low levels, which varied from 5 to 4,946,356. To better understand the role of miRNAs during the preservation of ponkan in vitro plantlet, the expression patterns of cre-miR156a/159b/160a/166a/167a/168a/171/398b were validated by quantitative real-time PCR (qPCR). The results showed that not only the development-associated miRNAs, e.g., cre-miR156/159/166/396, expressed highly at the early preservation period in the in vitro ponkan plantlet leaves but also the stress-related miRNAs, e.g., cre-miR171 and cre-miR398b, expressed highly at the same time. The expression levels of most tested miRNAs were found to decrease after 6 months and the amounts of these miRNAs were kept at low levels at 18 months. After analyzing the expression level of their targets during the reservation of the ponkan in vitro plantlet, development-associated cre-ARF6 and stress-related cre-CSD modules exhibited negative correlation with miR167 and miR398, respectively, indicating an involvement of the miRNAs in the in vitro development of ponkan and function in the conservation of ponkan germplasm.

Characterization and analysis of differentially expressed microRNAs in hircine ovaries during the follicular and luteal phases

Ovarian activity, which is mainly controlled by follicle-stimulating hormone and luteinizing hormone, is vital to successful reproduction and maintaining reproductive efficiency in livestock. To determine if the regulation of follicular-luteal transition occurs at the post-transcriptional level in hircine ovaries, the expression patterns of small RNAs in the ovarian tissues of Anhui white goats in the follicular and luteal phases were analyzed using Solexa sequencing. In total, 1039 miRNAs were co-expressed in the two libraries, and 278 and 469 miRNAs were specifically expressed in the hircine ovaries during the follicular and luteal phases, respectively. A total of 43 potential novel miRNAs were predicted in the two libraries. GO annotation and KEGG pathway analysis were applied to analyze the target genes of all miRNAs predicted in the two libraries. The highly and differentially expressed miRNAs included miR-26-5p, miR-145-5p, miR-145, miR-145a-5p, miR-125a-5p, miR-320d, and miR-320c, which may participate in follicular-luteal transition. Five co-expressed miRNAs, of which 2 were differentially expressed between the two libraries, were randomly selected to validate the expression pattern using RT-PCR, and the results were consistent with the Solexa sequencing data. Our present results help to clarify the roles of miRNAs in the regulation of follicular-luteal transition in goat ovaries, which may further enhance the reproductive efficiency of commercially important animals in the future.

De novo transcriptome analysis of tobacco seedlings and identification of the early response gene network under low-potassium stress.

Tobacco is an economically important crop, and its potassium content can greatly affect the quality of tobacco leaves. However, the molecular mechanism involved in potassium starvation in tobacco has not been elucidated to date. In this study, Illumina (Solexa) sequencing technology was used to analyze the transcriptome of tobacco seedlings under low-potassium stress for 6, 12, and 24 h. After analysis, 107,824 assembled unigenes were categorized into 57 GO functional groups, and 31,379 unigenes (29.08%) were clustered into 25 COG categories. A total of 9945 genes were classified into 233 KEGG pathways, and 15,209 SSRs were found among the 107,824 unigenes. Between the two samples, 1034 genes were differentially expressed. Twelve randomly selected gene expression levels were analyzed by quantitative RT-PCR, and the results were highly consistent with those obtained by Solexa sequencing. Our results provide a comprehensive analysis of the gene-regulatory network of tobacco seedlings under low-potassium stress.

A Group of Novel Serum Diagnostic Biomarkers for Multidrug-Resistant Tuberculosis by iTRAQ-2D LC-MS/MS and Solexa Sequencing.

The epidemic of pulmonary tuberculosis (TB), especially multidrug-resistance tuberculosis (MDR-TB) presented a major challenge for TB treatment today. We performed iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) and Solexa sequencing among MDR-TB patients, drug-sensitive tuberculosis (DS-TB) patients, and healthy controls. A total of 50 differentially expressed proteins and 43 differentially expressed miRNAs (fold change >1.50 or <0.60, P<0.05) were identified in the MDR-TB patients compared to both DS-TB patients and healthy controls. We found that 22.00% of differentially expressed proteins and 32.56% of differentially expressed miRNAs were related, and could construct a network mainly in complement and coagulation cascades. Significant differences in CD44 antigen (CD44), coagulation factor XI (F11), kininogen-1 (KNG1), miR-4433b-5p, miR-424-5p, and miR-199b-5p were found among MDR-TB patients, DS-TB patients and healthy controls (P<0.05) by enzyme-linked immunosorbent assay (ELISA) and SYBR green qRT-PCR validation. A strong negative correlation, consistent with the target gene prediction, was found between miR-199b-5p and KNG1 (r=-0.232, P=0.017). Moreover, we established the MDR-TB diagnostic model based on five biomarkers (CD44, KNG1, miR-4433b-5p, miR-424-5p, and miR-199b-5p). Our study proposes potential biomarkers for MDR-TB diagnosis, and also provides a new experimental basis to understand the pathogenesis of MDR-TB.

Transcriptome expression profiling of fur color formation in domestic rabbits using Solexa sequencing.

Fur color is an important, genetically determined characteristic of domestic rabbits, and rabbit furs are of great economic value. To investigate the molecular genetics associated with fur color determination in domestic rabbits, we used Solexa-sequencing technology to probe gene expression in dorsal skin tissues sampled from full-sibling Rex rabbits of different colors. The number of expressed genes in each sample was approximately 14,700. Among the top 30 genes and transcription factors with the highest reads per kilobase per million values, the elongation factor-alpha 1 gene was highly expressed in all samples, as were genes of the ribosomal protein and keratin gene families. Compared with the chinchilla (C) Rex rabbit control sample, the numbers of genes in the black (B) and white (W) rabbit samples were 1809 and 460, respectively, and the number of common differentially expressed genes was 257. Clustering analysis of these 257 genes revealed that 32 were up-regulated in sample B and down-regulated in sample W. Of these 32 genes, we identified some that are related to fur formation, including Tyrosinase-related protein 1 (TYRP1) and Tyrosinase (TYR), as well as genes with unknown functions. Quantitative real-time polymerase chain reaction was used to verify the expression patterns of those genes. The findings are expected to provide reference for the further study of fur color formation in rabbits.

Identification of miRNAs in sweet potato by solexa sequencing

Identification of miRNAs in sweet potato by solexa sequencing