Generation High-throughput Sequencing Technology Research Articles

Detection of TP53 gene mutation in blood of breast cancer patients based on circulating tumor DNA and its application in prognosis.

This experiment was carried out to explore the application value of high throughput gene sequencing technology in detecting TP53 gene mutations in the blood of patients with breast cancer by detecting ctDNA gene mutations, and exploring the relationship between TP53 mutations and clinicopathological characteristics and prognosis of patients. The gene mutation of peripheral blood ctDNA and tissue paraffin DNA (tDNA) of 50 patients was detected by high-throughput sequencing technology. The basic data of 50 cases of Medium to high-risk breast cancer diagnosed and were retrospectively collected, and the clinicopathological characteristics and survival results of TP53 mutant and wild-type patients were compared and analyzed according to the ctDNA detection results and relevant follow-up data. Analyze the impact of TP53 mutations on overall survival and progression-free survival using univariate and multivariate Cox regression models. Among 50 patients, there were 29 cases of 7 kinds of gene mutations detected by ctDNA, and 37 cases of 9 kinds of gene mutations detected by tDNA. Using the gene mutation results detected by tDNA as the gold standard, the sensitivity and specificity of peripheral blood ctDNA in diagnosing TP53 gene mutations are 75% to 100%, 92.31% to 100%, and the overall coincidence rate with tDNA results was 83.33% to 100%. Exon 5 was the most prone to mutation, with a frequency of 24.14% (7/29). The most common type of mutation was the missense mutation of 37.93% (11/29). There was no significant correlation between TP53 mutation and PFS (HR=0.67, 95% CI: 0.41-1.08, P=0.102), while TP53 mutation was a protective factor for OS (HR=0.49, 95% CI: 0.27-0.90, P=0.022). The detection of ctDNA in peripheral blood of breast cancer patients by high-throughput gene sequencing technology can replace tumor tissue sections to understand gene mutation. The TP53 mutation in breast cancer patients is related to tumor size, lymph node metastasis and vascular tumor thrombus, but the prognosis of TP53 mutant patients is similar to that of wild-type patients.

The Effect of Combining Millet and Corn Straw as Source Forage for Beef Cattle Diets on Ruminal Degradability and Fungal Community

Three ruminal cannulated Simmental crossbreed bulls (approximately 3 years of age and with 380 ± 20 kg live weight at initiation of the experiment) were used in a 3 × 3 Latin square experiment in order to determine the effects of the treatments on ruminal pH and degradability of nutrients, as well as the rumen fungal community. The experimental periods were 21 d, with 18 d of adjustment to the respective dietary treatments and 3 d of sample collection. Treatments consisted of a basal diet containing a 47.11% composition of two sources of forage as follows: (1) 100% millet straw (MILLSTR), (2) 50:50 millet straw and corn straw (COMB), and (3) 100% corn straw (CORNSTR). Dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) were tested for ruminal degradability using the nylon bag method, which was incubated for 6, 12, 24, 36, 48, and 72 h, and rumen fungal community in rumen fluid was determined by high-throughput gene sequencing technology. Ruminal pH was not affected by treatments. At 72 h, compared to MILLSTR, DM degradability of CORNSTR was 4.8% greater (p < 0.05), but when corn was combined with millet straw, the difference in DM degradability was 9.4%. During the first 24 h, degradability of CP was lower for CORNSTR, intermediate for MILLSTR, and higher for COMB. However, at 72 h, MILLSTR and COMB had a similar CP degradability value, staying greater than the CP degradability value of the CORNSTR treatment. Compared to MILLSTR, the rumen degradability of NDF was greater for CORNSTR and intermediate for the COMB. There was a greater degradability for ADF in CORNSTR, intermediate for COMB, and lower for MILLSTR. In all treatments, Ascomycota and Basidiomycota were dominant flora. Abundance of Basidiomycota in the group COMB was higher (p < 0.05) than that in the group CORNSTR at 12 h. Relative to the fungal genus level, the Thelebolus, Cladosporium, and Meyerozyma were the dominant fungus, and the abundance of Meyerozyma in COMB and CORNSTR were greater (p < 0.05) than MILLSTR at 12, 24, and 36 h of incubation. In conclusion, it is suggested to feed beef cattle with different proportions of millet straw and corn straw combinations.

Integrated Analysis of Transcriptome and microRNA Profile Reveals the Toxicity of Euphorbia Factors toward Human Colon Adenocarcinoma Cell Line Caco-2.

Euphorbia factors, lathyrane-type diterpenoids isolated from the medical herb Euphorbia lathyris L. (Euphorbiaceae), have been associated with intestinal irritation toxicity, but the mechanisms underlying this phenomenon are still unknown. The objective of this study was to evaluate the transcriptome and miRNA profiles of human colon adenocarcinoma Caco-2 cells in response to Euphorbia factors L1 (EFL1) and EFL2. Whole transcriptomes of mRNA and microRNA (miRNA) were obtained using second generation high-throughput sequencing technology in response to 200 μM EFL treatment for 72 h, and the differentially expressed genes and metabolism pathway were enriched. Gene structure changes were analyzed by comparing them with reference genome sequences. After 72 h of treatment, 16 miRNAs and 154 mRNAs were differently expressed between the EFL1 group and the control group, and 47 miRNAs and 1101 mRNAs were differentially expressed between the EFL2 group and the control. Using clusters of orthologous protein enrichment, the sequenced mRNAs were shown to be mainly involved in transcription, post-translational modification, protein turnover, chaperones, signal transduction mechanisms, intracellular trafficking, secretion, vesicular transport, and the cytoskeleton. The differentially expressed mRNA functions and pathways were enriched in transmembrane transport, T cell extravasation, the IL-17 signaling pathway, apoptosis, and the cell cycle. The differentially expressed miRNA EFLs caused changes in the structure of the gene, including alternative splicing, insertion and deletion, and single nucleotide polymorphisms. This study reveals the underlying mechanism responsible for the toxicity of EFLs in intestinal cells based on transcriptome and miRNA profiles of gene expression and structure.

Effect of Different Land Use Types on the Diversity of Soil Bacterial Community in the Coastal Zone of Jialing River

The purpose of this study was to clarify the differences in soil bacterial community diversity in different land uses in the coastal zone of the Jialing River and to provide theoretical reference for reasonable land development and resource protection of the Jialing River basin. Based on second generation high-throughput sequencing technology, the diversity, structural composition, functional types, and influencing factors of soil bacterial communities in different land use types were analyzed. The results showed that:① there were some differences in soil physical and chemical properties among different land uses. The nutritional indexes of agricultural land and soil wetland were significantly higher than those of natural forest land and sandy soil wetland (P<0.05). ②The Ace, Shannon, and Chao indexes of sandy soil wetland and soil wetland were significantly higher than those of natural forest land and agricultural land (P<0.05). The Simpson index of soil bacteria in different land use types was significantly different (P<0.05). ③ The dominant phyla of soil bacteria in the coastal zone of the Jialing River were Proteobacteria, Actinobacteriata, Acidobacteriata, and Chloroflexi, and the relative abundance accounted for more than 65%. The community structure of sandy soil wetland was similar to that of soil wetland and was significantly different from that of other land use types (P<0.05). ④ Primary functional metabolism was the core function of the bacterial community, accounting for more than 75%. Agricultural activities had the greatest impact on the community function, which significantly improved the metabolism of amino acids and carbohydrates, enhanced the utilization of nitrogen and phosphorus, reduced the metabolism of energy and coenzyme vitamins, and weakened the resistance of bacterial community to environmental impact (P<0.05). ⑤ Total phosphorus (TP) was the key influencing factor of the soil bacterial community. In conclusion, the construction of the wetland habitat in the Jialing River coastal zone is most conducive to the development of underground ecological diversity and the stability of the soil ecosystem.

Fungal community characteristics and driving factors during the decaying process of Salix psammophila sand barriers in the desert.

Wood-inhabiting fungi are crucial to wood decay and decomposition in S. psammophila sand barriers, which in turn consumingly influence nutrient dynamics in desert soils. In the case of an extremely arid desert, as opposed to forests, little of known about the fungal community composition of decaying wood and the effects of decomposing wood on soil physical and chemical properties. Combined with high-throughput gene sequencing technology, we investigated the relationships between microenvironment factors with fungal community composition and diversity during the decomposition of Salix psammophila sand barriers. The results showed that the destruction of lignocellulose components during the decay process of S. psammophila sand barrier alters the physical and chemical properties of the surrounding soil. Compared with one-year sand barrier, lignin and cellulose of seven-year S. psammophila sand barrier decreased by 40.48% and 38.33%, respectively. Soil available potassium and available nitrogen increased by 39.80% and 99.46%, respectively. We confirmed that soil available nitrogen, soil pH and soil moisture content significantly affected the fungal community distribution of S. psammophila sand barriers. Sordariomycetes are mainly affected by the positive correlation of soil pH, while Eurotiomycetes are most affected by the positive correlation of soil moisture content and soil porosity. Although our results highlighted the importance of bidirectional interactions between fungi in decayed sand barriers and soil properties, their contribution to the desert ecosystem still needs further confirmation from future studies. However, overall our findings improved the current understanding of the sand barrier-soil interactions on the process of ecological restoration.

Application of transcriptome sequencing and fusion genes analysis in the diagnosis of myeloid leukemia with normal karyotype

Objective: To analyze the possible fusion genes with high-throughput transcriptome sequencing in myeloid leukemia patients with normal karyotype. Methods: From May 2017 to January 2019, three cases of myeloid leukemia patients with normal karyotype and negative for common fusion genes from the First Affiliated Hospital of Nanchang University were selected as the research objects. The transcriptome sequencing of bone marrow mononuclear cells was performed by high-throughput gene sequencing technology. Defuse software was used to analyze the gene fusion sequence in the transcriptome data, reverse-transcription polymerase chain reaction (RT-PCR) and Sanger sequencing were used to verify the fusion gene with clear pathological significance. Results: All three patients were diagnosed with myeloid leukemia by clinical manifestations, bone marrow cell morphology, immunology, and histochemical staining. Cytogenetic tests showed normal chromosome karyotypes. Fluorescence in situ hybridization and RT-PCR were used to detect BCR-ABL1, PML-RARA, and other common fusion genes. The results were all negative. Transcriptome sequencing and fusion transcripts analysis revealed that these three patients carried rare fusion genes with clear pathological significance, which included BCR-FGFR1, CPSF6-RARG, and NUP98-RARG, respectively. Conclusion: Transcriptome sequencing can accurately analyze rare but pathologically significant fusion genes that may exist in myeloid leukemia patients with normal karyotypes.

Aspergillosis infection over 20\u2009years: a case report of probable vascular invasion in central nervous system

BackgroundAspergillosis infection of central nervous system (CNS) is rare and fatal. Diagnosis of invasive aspergillosis remains difficult. Aspergillosis of CNS can be an acute, subacute, or chronic onset, and the longest course of the disease was currently reported to be 4 years. Here, we report a case with recurrent headache over 20 years.Case presentationA 54-year-old man was admitted to our neurological disease ward due to intermittent throbbing headache lasting for more than 20 years that had grown notably worse over the past week. The headache was localized to the side of his head blown by a cold wind. He also experienced nausea and vomiting when the headache became severe. The headache usually lasted for 3–4 h after he was blown by the cold wind, though he had no fever. The neurological examination was normal. Magnetic resonance imaging (MRI) of the brain was negative for parenchymal and meningeal lesions. However, the case had increased intracranial pressure (ICP), and elevated protein level in the cerebrospinal fluid (CSF). Aspergillus fumigatus was found in CSF by nanopore targeted sequencing (NTS) and in blood by enzyme-linked immunosorbent assay (ELISA). Aspergillus fumigatus-specific antibody IgG was 104.62 AU/mL, aspergillus galactomannan (GM) antigen was 3.27 μg/L, D-dimer was 3.25 mg/L and fibrinogen degradation product was 11.50 mg/L, which were markedly higher than the normal levels. The patient was prescribed by voriconazole. After the treatment of 14 days, the ICP, CSF protein level, Aspergillus fumigatus-specific antibody IgG, GM antigen, D-dimer and fibrinogen degradation product returned normal. Aspergillus was disappeared by NTS test of CSF. His headache has never occurred again after blowing by a cold wind.ConclusionsThis report reveals that aspergillosis infection of CNS can last for more than 20 years and the major symptom is only intermittent throbbing headache in an immunocompetent patient. Vascular invasion is probably the mechanism of headache in our case with CNS aspergillosis infection. Performing high-throughput gene sequencing technology in CSF is important when the pathogen is not determined for the patients with suspected CNS infection.

Application progress of transcriptome sequencing in hematological malignancies

Transcriptome sequencing (RNA-seq) has unique advantages in analyzing gene fusion, splicing mutations, and gene expression profiles. Single-cell RNA-seq provides powerful tools to reveal cellular heterogeneity in normal and tumor tissues. With the widespread application of high-throughput gene sequencing technology and the rapid reduction in cost, RNA-seq is increasingly used in hematological malignancies research. This article introduces the related research progress in conjunction with reports at the 61st American Society of Hematology Annual Meeting. Key words: High-throughput nucleotide sequencing; Transcriptome; Gene sequencing; Fusion genes; Gene mutations; Hematological malignancies

High-Throughput Sequencing of Plasma Free DNA In the Second Trimester for Non-Invasive Prenatal Testing of 21-Trisomy Syndrome

Objective: To investigate the application of fetal free DNA (cff DNA) in the peripheral blood of pregnant women to the diagnosis of non-invasive prenatal bowel syndrome (21-trisomy). Methods: 1735 cases of single-pregnant pregnant women with 12-24 weeks of pregnancy were collected, and peripheral blood DNA was extracted. The new generation high-throughput sequencing technology was used for detection and bioinformatics analysis, and amniotic fluid or umbilical cord was selected for 21-three high-risk pregnant women. Blood karyotype analysis was performed. Result: In the 1735 specimens, 12 cases of 21-trisomy positive samples were detected by the new non-invasive prenatal technique, and 12 cases were confirmed by karyotype analysis. The sensitivity and specificity of the non-invasive prenatal technique for the 21-trisomy detection were 100%. Conclusion: The clinical non-invasive prenatal 21-trisomy diagnosis of cffDNA has broad application prospects in prenatal screening.

A Multireference-Based Whole Genome Assembly for the Obligate Ant-Following Antbird, Rhegmatorhina melanosticta (Thamnophilidae)

Current generation high-throughput sequencing technology has facilitated the generation of more genomic-scale data than ever before, thus greatly improving our understanding of avian biology across a range of disciplines. Recent developments in linked-read sequencing (Chromium 10×) and reference-based whole-genome assembly offer an exciting prospect of more accessible chromosome-level genome sequencing in the near future. We sequenced and assembled a genome of the Hairy-crested Antbird (Rhegmatorhina melanosticta), which represents the first publicly available genome for any antbird (Thamnophilidae). Our objectives were to (1) assemble scaffolds to chromosome level based on multiple reference genomes, and report on differences relative to other genomes, (2) assess genome completeness and compare content to other related genomes, and (3) assess the suitability of linked-read sequencing technology for future studies in comparative phylogenomics and population genomics studies. Our R. melanosticta assembly was both highly contiguous (de novo scaffold N50 = 3.3 Mb, reference based N50 = 53.3 Mb) and relatively complete (contained close to 90% of evolutionarily conserved single-copy avian genes and known tetrapod ultraconserved elements). The high contiguity and completeness of this assembly enabled the genome to be successfully mapped to the chromosome level, which uncovered a consistent structural difference between R. melanosticta and other avian genomes. Our results are consistent with the observation that avian genomes are structurally conserved. Additionally, our results demonstrate the utility of linked-read sequencing for non-model genomics. Finally, we demonstrate the value of our R. melanosticta genome for future researchers by mapping reduced representation sequencing data, and by accurately reconstructing the phylogenetic relationships among a sample of thamnophilid species.

Whole Genome Sequencing and Analysis of Chlorimuron-Ethyl Degrading Bacteria Klebsiella pneumoniae 2N3.

Klebsiella pneumoniae 2N3 is a strain of gram-negative bacteria that can degrade chlorimuron-ethyl and grow with chlorimuron-ethyl as the sole nitrogen source. The complete genome of Klebsiella pneumoniae 2N3 was sequenced using third generation high-throughput DNA sequencing technology. The genomic size of strain 2N3 was 5.32 Mb with a GC content of 57.33% and a total of 5156 coding genes and 112 non-coding RNAs predicted. Two hydrolases expressed by open reading frames (ORFs) 0934 and 0492 were predicted and experimentally confirmed by gene knockout to be involved in the degradation of chlorimuron-ethyl. Strains of ΔORF 0934, ΔORF 0492, and wild type (WT) reached their highest growth rates after 8–10 hours in incubation. The degradation rates of chlorimuron-ethyl by both ΔORF 0934 and ΔORF 0492 decreased in comparison to the WT during the first 8 hours in culture by 25.60% and 24.74%, respectively, while strains ΔORF 0934, ΔORF 0492, and the WT reached the highest degradation rates of chlorimuron-ethyl in 36 hours of 74.56%, 90.53%, and 95.06%, respectively. This study provides scientific evidence to support the application of Klebsiella pneumoniae 2N3 in bioremediation to control environmental pollution.

Use of comparative transcriptome analysis to identify candidate genes related to albinism in channel catfish (Ictalurus punctatus)

Transcriptome profiles of albino and wild type catfish were generated using next generation high-throughput RNA sequencing technology and compared. A total of 1106 differentially expressed genes were obtained, of which 530 were up-regulated and 576 were down-regulated in the skin tissue of albino catfish relative to wild type catfish. In total, 30,813 alternative splicing (AS) events were identified. These AS events were distributed in 15,090 genes with an average of 2.04 AS events per gene. We identified and compared a large proportion of transcripts encoding genes involved in regulation of melanogenesis, melanosome biogenesis, melanosome transport, and the tyrosine metabolism pathway in the catfish skin transcriptome dataset. Further analysis revealed that differentially expressed genes, including MART1, Cdk2, PMEL17, Tyr, Tyrp1, ADH, HGD, and TYRL, were significantly overrepresented in the melanogenesis pathway and in several biological processes associated with pigmentation. The albino catfish skin samples exhibited significantly increased expression levels of all candidate genes in the tyrosine metabolism pathway. We also identified a possible mechanism for the mutation of the catfish Hps4 gene, which affects regulation of expression of genes in the tyrosine metabolism pathway. Our findings provide a theoretical basis for further understanding of the pathogenesis of albinism in catfish.

Nanopore sequencing and full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations

BackgroundHuman cytomegalovirus (HCMV) has a double-stranded DNA genome of approximately 235 Kbp that is structurally complex including extended GC-rich repeated regions. Genomic recombination events are frequent in HCMV cultures but have also been observed in vivo. Thus, the assembly of HCMV whole genomes from technologies producing shorter than 500 bp sequences is technically challenging. Here we improved the reconstruction of HCMV full genomes by means of a hybrid, de novo genome-assembly bioinformatics pipeline upon data generated from the recently released MinION MkI B sequencer from Oxford Nanopore Technologies.ResultsThe MinION run of the HCMV (strain TB40/E) library resulted in ~ 47,000 reads from a single R9 flowcell and in ~ 100× average read depth across the virus genome. We developed a novel, self-correcting bioinformatics algorithm to assemble the pooled HCMV genomes in three stages. In the first stage of the bioinformatics algorithm, long contigs (N50 = 21,892) of lower accuracy were reconstructed. In the second stage, short contigs (N50 = 5686) of higher accuracy were assembled, while in the final stage the high quality contigs served as template for the correction of the longer contigs resulting in a high-accuracy, full genome assembly (N50 = 41,056). We were able to reconstruct a single representative haplotype without employing any scaffolding steps. The majority (98.8%) of the genomic features from the reference strain were accurately annotated on this full genome construct. Our method also allowed the detection of multiple alternative sub-genomic fragments and non-canonical structures suggesting rearrangement events between the unique (UL /US) and the repeated (T/IRL/S) genomic regions.ConclusionsThird generation high-throughput sequencing technologies can accurately reconstruct full-length HCMV genomes including their low-complexity and highly repetitive regions. Full-length HCMV genomes could prove crucial in understanding the genetic determinants and viral evolution underpinning drug resistance, virulence and pathogenesis.

Fingerprint of Exhaust Gases and Database of Microbial Diversity During Silkworm Excrement Composting

ABSTRACTComposting can convert silkworm excrement (SE) into good fertilizer, but the popularization of this practice is greatly hampered due to the strong odor produced. Determining the gas pollutants, the potential native aerogenic microorganisms and deodorant microorganism communities will lay foundations for developing good strategies to deal with malodor pollution from SE composting. This study qualitatively and quantitatively analyzed the exhaust gas and the microbial diversity. With a combination of solid-phase micro-extraction-gas chromatography tandem mass spectrometry technology and other methods of Chinese national standards, 43 odor pollutants from SE composting were identified and quantified. Ammonia, sulfur dioxide and hydrogen sulfide were the three major pollutants among the volatile inorganic compounds, with concentrations far exceeding the permissible standards for workplaces in China. 5-Ethyl-2,2,3-trimethyl-heptane, 2,2,4,6,6-5-methyl-heptane and 5-isobutyl-nonane accounted for 46.58% of volatile organic compounds. Using analysis of ribosome DNA sequences with Illumina Miseq PE 2 * 250, a next generation high-throughput sequencing technology, more than 694 bacteria (and actinomycetes) and more than 136 fungi were annotated, including groups of native aerogenic microorganisms and deodorant microorganisms. This study indicated a serious malodor problem and abundant microorganisms from SE composting and provided basic data not only for odor pollution control but also for future functional microorganism studies.

Methods for Identification of Protein-RNA Interaction.

The importance of RNA-protein interactions in regulation of mRNA and non-coding RNA function is increasingly appreciated. With the development of next generation high-throughput sequencing technologies, a variety of methodshave been proposed to comprehensively identify RNA-protein interactions. In this chapter, we discussed the traditional and state-of-the-art technologies that were used to study RNA-protein interaction, including experimental and computational methods. To help highlight the biological significance of RNA-protein interaction in complex diseases, online resources on RNA-protein interactions were briefly discussed. Finally, we discussed the interaction among noncoding RNAs (such as long noncoding RNAs and microRNAs) and proteins, as well as the dysregulation of RNA-protein interaction in complex diseases. These summarization will ultimately provide a more complete picture for understanding of the function of RNA-protein interactions, including how these interaction assembled and how they modulate cellular function in complex diseases.

Characterization of a de novo assembled transcriptome of the Common Blackbird (Turdus merula).

BackgroundIn recent years, next generation high throughput sequencing technologies have proven to be useful tools for investigations concerning the genomics or transcriptomics also of non-model species. Consequently, ornithologists have adopted these technologies and the respective bioinformatics tools to survey the genomes and transcriptomes of a few avian non-model species. The Common Blackbird is one of the most common bird species living in European cities, which has successfully colonized urban areas and for which no reference genome or transcriptome is publicly available. However, to target questions like genome wide gene expression analysis, a reference genome or transcriptome is needed.MethodsTherefore, in this study two Common Blackbirds were sacrificed, their mRNA was isolated and analyzed by RNA-Seq to de novo assemble a transcriptome and characterize it. Illumina reads (125 bp paired-end) and a Velvet/Oases pipeline led to 162,158 transcripts. For the annotation (using Blast+), an unfiltered protein database was used. SNPs were identified using SAMtools and BCFtools. Furthermore, mRNA from three single tissues (brain, heart and liver) of the same two Common Blackbirds were sequenced by Illumina (75 bp single-end reads). The draft transcriptome and the three single tissues were compared by their BLAST hits with the package VennDiagram in R.ResultsFollowing the annotation against protein databases, we found evidence for 15,580 genes in the transcriptome (all well characterized hits after annotation). On 18% of the assembled transcripts, 144,742 SNPs were identified which are, consequently, 0.09% of all nucleotides in the assembled transcriptome. In the transcriptome and in the single tissues (brain, heart and liver), 10,182 shared genes were found.DiscussionUsing a next-generation technology and bioinformatics tools, we made a first step towards the genomic investigation of the Common Blackbird. The de novo assembled transcriptome is usable for downstream analyses such as differential gene expression analysis and SNP identification. This study shows the importance of the approach to sequence single tissues to understand functions of tissues, proteins and the phenotype.

Decreased miRNA expression in Klinefelter syndrome

The widelyvariable phenotypic spectrum and the different severity of symptoms in men with Klinefelter syndrome (KS) suggest a role for epigenetic mediators. Therefore, the aim of this study is to evaluate the possible involvement of miRNAs in the clinical manifestations of KS. To accomplish this, we performed a transcriptome analysis in peripheral blood mononuclear cells (PBMCs) of 10 non-mosaic KS patients, 10 aged-matched healthy men and 10 aged-matched healthy female controls with normal karyotype. After RNA extraction from PBMC and the preparation of RNA libraries, the samples were sequenced using next generation high-throughput sequencing technology. Expression profiling analysis revealed a significant differential expression of 2 miRNAs in KS compared to male controls. In particular, MIR3648 resulted significantly (q-value < 0.0001) down-regulated by −19.084- fold, while MIR3687was strongly down-regulated (q-value < 0.0001) considering KS patients. These results were confirmed by qRT-PCR. The functional analysis of the two transcripts showed that they seem to play a role in breast cancer, hemopoietic abnormalities, immune defects and adipocyte differentiation and fat cell maturation. Therefore, we speculate that both miRNAs may play a role in the immune and metabolic disorders and in the risk of breast cancer development in men with KS.

High throughput deep sequencing reveals the important roles of microRNAs during sweetpotato storage at chilling temperature

Sweetpotato (Impomoea batatas L.) is a globally important economic food crop with a potential of becoming a bioenergy and pharmaceutical crop. Thus, studying the molecular mechanism of tuberous root development and storage is very important. However, not too much progress has been made in this field. In this study, we employed the next generation high-throughput deep sequencing technology to sequence all small RNAs and degradome of sweetpotato for systematically investigating sweetpotato response to chilling stress during storage. A total of 190 known microRNAs (miRNAs) and 191 novel miRNAs were identified, and 428 transcripts were targeted by 184 identified miRNAs. More importantly, we identified 26 miRNAs differentially expressed between chilling stress and control conditions. The expression of these miRNAs and their targets was also confirmed by qRT-PCR. Integrated analysis of small RNAs and degradome sequencing reveals that miRNA-mediated SA signaling, ABA-dependent, and ROS response pathways are involved in sweetpotato root response to chilling stress during storage.

Integrative Network Analysis of Newly Diagnosed Multiple Myeloma Identifies a Novel RNA-Seq Based High Riskgene Signature

High-risk Multiple Myeloma (MM) is characterized by unresponsiveness to multiple therapies, rapid disease progression and short overall survival, and may be significantly different from relapsed MM, where aggressiveness is usually a result of drug-resistance associated to clonal selection. Several gene expression-based signatures have been proposed in the past years, however the identification of high-risk patients at diagnosis still represents a challenge. Next generation high-throughput sequencing technologies have enabled a deeper insight into cancer genomes and transcriptomes at an unprecedented level of detail. MMRF CoMMpass is a longitudinal, prospective observational study, started in 2011, that aims to collect and analyze sequencing and clinical data from >1,000 MM patients at initial diagnosis and at relapse. CoMMpass is a real world observational study and, as such, reflects the therapeutic heterogeneity seen across patient populations and provides a unique opportunity to correlate molecular profiles, genomic alterations and clinical characteristics of MM with treatment outcome.Here we present a network approach to identify high-risk myeloma patients developed using next generation sequencing data from 450 patients in the IA7 release of CoMMpass. We generated MMNet, an integrated network model of newly diagnosed myeloma based on RNA-seq, Whole-Exome (WXS) and Whole-Genome (WGS) data correlated with clinical outcomes. MMNet consisted of 37 modules of coexpressed genes, that were further characterized by functional enrichment analysis and correlation with clinical traits and genomic alterations, i.e. somatic mutations and copy number alterations inferred from WGS and WXS data.A total of 89 progression/death events have been reported for the cohort within the second year since the beginning of the study. Cox regression analysis identified a module of co-expressed genes whose over-expression was significantly correlated with early relapse (<2yr) (HR 1.75, 95%CI = 1.169-2.614, p=0.005). The module was also associated to stage III R-ISS, high clonality (>4 clones) and high mutational burden, as well as higher percentage of plasma cells in both bone marrow and peripheral blood, which are traits associated with high-risk disease. Module expression was also up regulated in patients with mutations in TP53 and MAX, 13q deletion and 1q amplification.We further narrowed down the signature to 286 genes (the MMNet-286 signature) strongly correlated with time to Event Free Survival (EFS) (r = -0.81, p = 0). This gene-set was significantly enriched for several pathways including Cell Cycle, DNA repair and Homologous Recombination (q < 0.01). Cox regression analysis showed that the two clusters induced by MMNet-286 discriminated between lower and higher risk patients with respect to EFS (HR = 2.22, 95% CI = 1.505-3.295, p = 4.007e-5) (Fig. 1). The prognostic value of MMNet-286 was confirmed on two independent datasets: Broyl-2010 (HR = 1.76, 95% CI = 1.182-2.642, p = 0.005) and Shaughnessy-2006 (HR = 2.65, 95% CI = 1.746-4.031, p = 2.03e-6) (Fig. 2 and 3). The Broyl-2010 dataset consisted of 275 samples from newly diagnosed myeloma patients included in the HOVON65/GMMG-HD4 trial (GSE19784). The Shaughnessy-2006 dataset consisted of 559 samples from newly diagnosed patients pre-TT2 and -TT3 treatments (GSE2658). Comparison of MMNet-286 with previous high risk signatures and disease classes revealed an overlap of five genes with the UAMS-70 signature, twelve genes with the EMC-92 signature and fifteen genes with the set of up-regulated genes in the UAMS PR class, for which the coexpression module was enriched.In Conclusion, our results demonstrate the advantages of employing integrated network models to identify prognostic features based on next generation sequencing data from large cohort of patients. Applications of the MMNet-286 signature include the generation of a prognostic assay (i.e. NanoString) for the identification of high-risk patients. Future work will aim at validation of the signature in larger cohorts from CoMMpass and other studies. [Display omitted] [Display omitted] [Display omitted] DisclosuresChari:Novartis: Consultancy, Research Funding; Array Biopharma: Consultancy, Research Funding; Pharmacyclics: Research Funding; Amgen Inc.: Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding. Cho:Genentech Roche: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Agenus, Inc.: Research Funding; Ludwig Institute for Cancer Research: Membership on an entity’s Board of Directors or advisory committees; Janssen: Consultancy, Research Funding. Barlogie:Signal Genetics: Patents & Royalties. Dudley:GlaxoSmithKline: Consultancy; Janssen Pharmaceuticals, Inc.: Consultancy; Ayasdi, Inc.: Equity Ownership; Ecoeos, Inc.: Equity Ownership; NuMedii, Inc.: Equity Ownership; Ontomics, Inc.: Equity Ownership; AstraZeneca: Speakers Bureau; NuMedii, Inc.: Patents & Royalties; Personalis: Patents & Royalties.

High-Throughput Sequencing Reveals H2O2 Stress-Associated MicroRNAs and a Potential Regulatory Network in Brachypodium distachyon Seedlings.

Oxidative stress in plants can be triggered by many environmental stress factors, such as drought and salinity. Brachypodium distachyon is a model organism for the study of biofuel plants and crops, such as wheat. Although recent studies have found many oxidative stress response-related proteins, the mechanism of microRNA (miRNA)-mediated oxidative stress response is still unclear. Using next generation high-throughput sequencing technology, the small RNAs were sequenced from the model plant B. distachyon 21 (Bd21) under H2O2 stress and normal growth conditions. In total, 144 known B. distachyon miRNAs and 221 potential new miRNAs were identified. Further analysis of potential new miRNAs suggested that 36 could be clustered into known miRNA families, while the remaining 185 were identified as B. distachyon-specific new miRNAs. Differential analysis of miRNAs from the normal and H2O2 stress libraries identified 31 known and 30 new H2O2 stress responsive miRNAs. The expression patterns of seven representative miRNAs were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, which produced results consistent with those of the deep sequencing method. Moreover, we also performed RT-qPCR analysis to verify the expression levels of 13 target genes and the cleavage site of 5 target genes by known or novel miRNAs were validated experimentally by 5′ RACE. Additionally, a miRNA-mediated gene regulatory network for H2O2 stress response was constructed. Our study identifies a set of H2O2-responsive miRNAs and their target genes and reveals the mechanism of oxidative stress response and defense at the post-transcriptional regulatory level.