Distribution of miRNA expression across human tissues.

We present a human miRNA tissue atlas by determining the abundance of 1997 miRNAs in 61 tissue biopsies of different organs from two individuals collected post-mortem. One thousand three hundred sixty-four miRNAs were discovered in at least one tissue, 143 were present in each tissue. To define the distribution of miRNAs, we utilized a tissue specificity index (TSI). The majority of miRNAs (82.9%) fell in a middle TSI range i.e. were neither specific for single tissues (TSI > 0.85) nor housekeeping miRNAs (TSI < 0.5). Nonetheless, we observed many different miRNAs and miRNA families that were predominantly expressed in certain tissues. Clustering of miRNA abundances revealed that tissues like several areas of the brain clustered together. Considering -3p and -5p mature forms we observed miR-150 with different tissue specificity. Analysis of additional lung and prostate biopsies indicated that inter-organism variability was significantly lower than inter-organ variability. Tissue-specific differences between the miRNA patterns appeared not to be significantly altered by storage as shown for heart and lung tissue. MiRNAs TSI values of human tissues were significantly (P = 10(-8)) correlated with those of rats; miRNAs that were highly abundant in certain human tissues were likewise abundant in according rat tissues. We implemented a web-based repository enabling scientists to access and browse the data (https://ccb-web.cs.uni-saarland.de/tissueatlas).

Validating Alzheimer's disease micro RNAs using next-generation sequencing.

INTRODUCTION: Molecular biomarkers for Alzheimer's disease (AD) can support detection and improved care for patients, but novel candidates require verification. We previously reported a 12-micro RNA (miRNA) blood-based signature using next-generation sequencing (NGS) of 54 AD cases and 22 controls. METHODS: We performed validation of 49 AD cases and 55 controls using NGS and also included 20 mild cognitive impairment and 90 multiple sclerosis samples to identify nonspecific markers. Thus, 103 AD cases, 77 unaffected controls, and 110 diseased controls were sequenced. Although the initial cohort came predominantly from the United States, the validation samples were collected in Germany. RESULTS: Five hundred eighty miRNAs were detected in the blood. In the initial cohort, we observed 203, in the validation cohort, 146 dysregulated miRNAs at a significance level of 0.05. With 68 miRNAs, the overlap was significant (P = .0003). Likewise, the area under the receiver operator characteristic curve values of the miRNAs correlated (correlation of 0.93; 95% confidence interval 0.89-0.96; P <10(-16)). DISCUSSION: MiRNAs have the potential to support AD diagnosis and patient care.

Double-Stranded Ligation Assay for the Rapid Multiplex Quantification of MicroRNAs.

MicroRNAs are auspicious candidates for a new generation of biomarkers. The detection of microRNA panels in body fluids promises early diagnosis of many diseases, including cancer or acute coronary syndrome. For a fast, sensitive, and specific detection of microRNA panels on the bedside, medical point-of-care systems that measure those biomarkers are required. As microchips are promising technical tools for a robust signal measurement at biochemical interfaces, we developed an assay for the electrochemical multiplex quantification of microRNAs on a CMOS chip with interdigitated gold electrode sensor positions. The method is based on the formation of a tripartite hybridization complex and subsequent both-sided ligation of the target nucleic acid to a reporter and capture strand. With a time to results of 30 min, the reported assay achieves a limit of detection below 1 pM, at a specificity down to single mismatch discrimination. It also offers very good signal dynamics between 1 pM and 1 nM, thus, allowing reliable quantification of the detected microRNAs and easy implementation into automated devices due to a simple workflow.

Influence of next-generation sequencing and storage conditions on miRNA patterns generated from PAXgene blood.

Whole blood derived miRNA signatures determined by Next-Generation Sequencing (NGS) offer themselves as future minimally invasive biomarkers for various human diseases. The PAXgene system is a commonly used blood storage system for miRNA analysis. Central to all miRNA analyses that aim to identify disease specific miRNA signatures, is the question of stability and variability of the miRNA profiles that are generated by NGS. We characterized the influence of five different conditions on the genome wide miRNA expression pattern of human blood isolated in PAXgene RNA tubes. In detail, we analyzed 15 miRNomes from three individuals. The blood was subjected to different numbers of freeze/thaw cycles and analyzed for the influence of storage at -80 or 8 °C. We also determined the influence of blood collection and NGS preparations on the miRNA pattern isolated from a single individual, which has been sequenced 10 times. Here, five PAXGene tubes were consecutively collected that have been split in two replicates, representing two experimental batches. All samples were analyzed by Illumina NGS. For each sample, approximately 20 million NGS reads have been generated. Hierarchical clustering and Principal Component Analysis (PCA) showed an influence of the different conditions on the miRNA patterns. The effects of the different conditions on miRNA abundance are, however, smaller than the differences that are due to interindividual variability. We also found evidence for an influence of the NGS measurement on the miRNA pattern. Specifically, hsa-miR-1271-5p and hsa-miR-182-5p showed coefficients of variation above 100% indicating a strong influence of the NGS protocol on the abundance of these miRNAs.

Influence of the confounding factors age and sex on microRNA profiles from peripheral blood.

BACKGROUND: MicroRNAs (miRNAs) measured from blood samples are promising minimally invasive biomarker candidates that have been extensively studied in several case-control studies. However, the influence of age and sex as confounding variables remains largely unknown. METHODS: We systematically explored the impact of age and sex on miRNAs in a cohort of 109 physiologically unaffected individuals whose blood was characterized by microarray technology (stage 1). We also investigated an independent cohort from a different institution consisting of 58 physiologically unaffected individuals having a similar mean age but with a smaller age distribution. These samples were measured by use of high-throughput sequencing (stage 2). RESULTS: We detected 318 miRNAs that were significantly correlated with age in stage 1 and, after adjustment for multiple testing of 35 miRNAs, remained statistically significant. Regarding sex, 144 miRNAs showed significant dysregulation. Here, no miRNA remained significant after adjustment for multiple testing. In the high-throughput datasets of stage 2, we generally observed a smaller number of significant associations, mainly as an effect of the smaller cohort size and age distribution. Nevertheless, we found 7 miRNAs that were correlated with age, of which 5 were concordant with stage 1. CONCLUSIONS: The age distribution of individuals recruited for case-control studies needs to be carefully considered, whereas sex may be less confounding. To support the translation of miRNAs into clinical application, we offer a web-based application (http://www.ccb.uni-saarland.de/mirnacon) to test individual miRNAs or miRNA signatures for their likelihood of being influenced.

Comprehensive analysis of microRNA profiles in multiple sclerosis including next-generation sequencing.

BACKGROUND: MicroRNAs (miRNAs) are short, noncoding RNAs with gene regulatory functions whose expression profiles may serve as disease biomarkers. OBJECTIVE: The objective of this study was to perform a comprehensive analysis of miRNA expression profiles in blood of patients with a clinically isolated syndrome (CIS) or relapsing-remitting multiple sclerosis (RRMS) including next-generation sequencing (NGS). METHODS: miRNA expression was analyzed in whole blood samples from treatment-naïve patients with CIS (n = 25) or RRMS (n = 25) and 50 healthy controls by NGS, microarray analysis, and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: In patients with CIS/RRMS, NGS and microarray analysis identified 38 and eight significantly deregulated miRNAs, respectively. Three of these miRNAs were found to be significantly up- (hsa-miR-16-2-3p) or downregulated (hsa-miR-20a-5p, hsa-miR-7-1-3p) by both methods. Another five of the miRNAs significantly deregulated in the NGS screen showed the same direction of regulation in the microarray analysis. qRT-PCR confirmed the direction of regulation for all eight and was significant for three miRNAs. CONCLUSIONS: This study identifies a set of miRNAs deregulated in CIS/RRMS and reconfirms the previously reported underexpression of hsa-miR-20a-5p in MS. hsa-miR-20a-5p and the other validated miRNAs may represent promising candidates for future evaluation as biomarkers for MS and could be of relevance in the pathophysiology of this disease.

miRTrail--a comprehensive webserver for analyzing gene and miRNA patterns to enhance the understanding of regulatory mechanisms in diseases.

BACKGROUND: Expression profiling provides new insights into regulatory and metabolic processes and in particular into pathogenic mechanisms associated with diseases. Besides genes, non-coding transcripts as microRNAs (miRNAs) gained increasing relevance in the last decade. To understand the regulatory processes of miRNAs on genes, integrative computer-aided approaches are essential, especially in the light of complex human diseases as cancer. RESULTS: Here, we present miRTrail, an integrative tool that allows for performing comprehensive analyses of interactions of genes and miRNAs based on expression profiles. The integrated analysis of mRNA and miRNA data should generate more robust and reliable results on deregulated pathogenic processes and may also offer novel insights into the regulatory interactions between miRNAs and genes. Our web-server excels in carrying out gene sets analysis, analysis of miRNA sets as well as the combination of both in a systems biology approach. To this end, miRTrail integrates information on 20.000 genes, almost 1.000 miRNAs, and roughly 280.000 putative interactions, for Homo sapiens and accordingly for Mus musculus and Danio rerio. The well-established, classical Chi-squared test is one of the central techniques of our tool for the joint consideration of miRNAs and their targets. For interactively visualizing obtained results, it relies on the network analyzers and viewers BiNA or Cytoscape-web, also enabling direct access to relevant literature. We demonstrated the potential of miRTrail by applying our tool to mRNA and miRNA data of malignant melanoma. MiRTrail identified several deregulated miRNAs that target deregulated mRNAs including miRNAs hsa-miR-23b and hsa-miR-223, which target the highest numbers of deregulated mRNAs and regulate the pathway "basal cell carcinoma". In addition, both miRNAs target genes like PTCH1 and RASA1 that are involved in many oncogenic processes. CONCLUSIONS: The application on melanoma samples demonstrates that the miRTrail platform may open avenues for investigating the regulatory interactions between genes and miRNAs for a wide range of human diseases. Moreover, miRTrail cannot only be applied to microarray based expression profiles, but also to NGS-based transcriptomic data. The program is freely available as web-server at mirtrail.bioinf.uni-sb.de.

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing.

The lack of efficient high-throughput methods for enrichment of specific sequences from genomic DNA represents a key bottleneck in exploiting the enormous potential of next-generation sequencers. Such methods would allow for a systematic and targeted analysis of relevant genomic regions. Recent studies reported sequence enrichment using a hybridization step to specific DNA capture probes as a possible solution to the problem. However, so far no method has provided sufficient depths of coverage for reliable base calling over the entire target regions. We report a strategy to multiply the enrichment performance and consequently improve depth and breadth of coverage for desired target sequences by applying two iterative cycles of hybridization with microfluidic Geniom biochips. Using this strategy, we enriched and then sequenced the cancer-related genes BRCA1 and TP53 and a set of 1000 individual dbSNP regions of 500 bp using Illumina technology. We achieved overall enrichment factors of up to 1062-fold and average coverage depths of 470-fold. Combined with high coverage uniformity, this resulted in nearly complete consensus coverages with >86% of target region covered at 20-fold or higher. Analysis of SNP calling accuracies after enrichment revealed excellent concordance, with the reference sequence closely mirroring the previously reported performance of Illumina sequencing conducted without sequence enrichment.

Targeted high throughput sequencing of a cancer-related exome subset by specific sequence capture with a fully automated microarray platform.

Sequence capture methods for targeted next generation sequencing promise to massively reduce cost of genomics projects compared to untargeted sequencing. However, evaluated capture methods specifically dedicated to biologically relevant genomic regions are rare. Whole exome capture has been shown to be a powerful tool to discover the genetic origin of disease and provides a reduction in target size and thus calculative sequencing capacity of >90-fold compared to untargeted whole genome sequencing. For further cost reduction, a valuable complementing approach is the analysis of smaller, relevant gene subsets but involving large cohorts of samples. However, effective adjustment of target sizes and sample numbers is hampered by the limited scalability of enrichment systems. We report a highly scalable and automated method to capture a 480 Kb exome subset of 115 cancer-related genes using microfluidic DNA arrays. The arrays are adaptable from 125 Kb to 1 Mb target size and/or one to eight samples without barcoding strategies, representing a further 26 - 270-fold reduction of calculative sequencing capacity compared to whole exome sequencing. Illumina GAII analysis of a HapMap genome enriched for this exome subset revealed a completeness of >96%. Uniformity was such that >68% of exons had at least half the median depth of coverage. An analysis of reference SNPs revealed a sensitivity of up to 93% and a specificity of 98.2% or higher.

Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates.

Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data (11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the 11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including 18 main species spanning a time period of 30 years. Species and drug specific resistance patterns were observed including increased resistance rates for Acinetobacter baumannii to carbapenems and for Escherichia coli to fluoroquinolones. Species-level pan-genomes were constructed to reflect the genetic repertoire of the respective species, including conserved essential genes and known resistance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed to infer gene-drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base, a resource available for academic research use free of charge at https://gear-base.com.

Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling.

Here we describe a novel microarray platform that integrates all functions needed to perform any array-based experiment in a compact instrument on the researcher's laboratory benchtop. Oligonucle otide probes are synthesized in situ via a light- activated process within the channels of a three-dimensional microfluidic reaction carrier. Arrays can be designed and produced within hours according to the user's requirements. They are processed in a fully automatic workflow. We have characterized this new platform with regard to dynamic range, discrimination power, reproducibility and accuracy of biological results. The instrument detects sample RNAs present at a frequency of 1:100 000. Detection is quantitative over more than two orders of magnitude. Experiments on four identical arrays with 6398 features each revealed a mean coefficient of variation (CV) value of 0.09 for the 6398 unprocessed raw intensities indicating high reproducibility. In a more elaborate experiment targeting 1125 yeast genes from an unbiased selection, a mean CV of 0.11 on the fold change level was found. Analyzing the transcriptional response of yeast to osmotic shock, we found that biological data acquired on our platform are in good agreement with data from Affymetrix GeneChips, quantitative real-time PCR and--albeit somewhat less clearly--to data from spotted cDNA arrays obtained from the literature.

cPAS-based sequencing on the BGISEQ-500 to explore small non-coding RNAs.

BACKGROUND: We present the first sequencing data using the combinatorial probe-anchor synthesis (cPAS)-based BGISEQ-500 sequencer. Applying cPAS, we investigated the repertoire of human small non-coding RNAs and compared it to other techniques. RESULTS: Starting with repeated measurements of different specimens including solid tissues (brain and heart) and blood, we generated a median of 30.1 million reads per sample. 24.1 million mapped to the human genome and 23.3 million to the miRBase. Among six technical replicates of brain samples, we observed a median correlation of 0.98. Comparing BGISEQ-500 to HiSeq, we calculated a correlation of 0.75. The comparability to microarrays was similar for both BGISEQ-500 and HiSeq with the first one showing a correlation of 0.58 and the latter one correlation of 0.6. As for a potential bias in the detected expression distribution in blood cells, 98.6% of HiSeq reads versus 93.1% of BGISEQ-500 reads match to the 10 miRNAs with highest read count. After using miRDeep2 and employing stringent selection criteria for predicting new miRNAs, we detected 74 high-likely candidates in the cPAS sequencing reads prevalent in solid tissues and 36 candidates prevalent in blood. CONCLUSIONS: While there is apparently no ideal platform for all challenges of miRNome analyses, cPAS shows high technical reproducibility and supplements the hitherto available platforms.

Longitudinal study on circulating miRNAs in patients after lung cancer resection.

There is an urgent need of comprehensive longitudinal analyses of circulating miRNA patterns to identify dynamic changes of miRNAs in cancer patients after surgery. Here we provide longitudinal analysis of 1,205 miRNAs in plasma samples of 26 patients after lung cancer resection at 8 time points over a period of 18 months and compare them to 12 control patients. First, we report longitudinal changes with respect to the number of detected miRNAs over time and identified a significantly increased number of miRNAs in patients developing metastases (p = 0.0096). A quantitative analysis with respect to the expression level of the detected miRNAs revealed more significant changes in the miRNA levels in samples from patients without metastases compared to the non-cancer control patients. This analysis provided further evidence of miRNA plasma levels that are changing over time after tumor resection and correlate to patient outcome. Especially hsa-miR-197 could be validated by qRT-PCR as prognostic marker. Also for this miRNA, patients developing metastases had levels close to that of controls while patients that did not develop metastases showed a significant up-regulation.In conclusion, our data indicate that the overall miRNome of a patient that later develops metastases is less affected by surgery than the miRNome of a patient who does not show metastases. The relationship between altered plasma levels of specific miRNAs with the development of metastases would partially have gone undetected by an analysis at a single time point only.

A blood based 12-miRNA signature of Alzheimer disease patients.

BACKGROUND: Alzheimer disease (AD) is the most common form of dementia but the identification of reliable, early and non-invasive biomarkers remains a major challenge. We present a novel miRNA-based signature for detecting AD from blood samples. RESULTS: We apply next-generation sequencing to miRNAs from blood samples of 48 AD patients and 22 unaffected controls, yielding a total of 140 unique mature miRNAs with significantly changed expression levels. Of these, 82 have higher and 58 have lower abundance in AD patient samples. We selected a panel of 12 miRNAs for an RT-qPCR analysis on a larger cohort of 202 samples, comprising not only AD patients and healthy controls but also patients with other CNS illnesses. These included mild cognitive impairment, which is assumed to represent a transitional period before the development of AD, as well as multiple sclerosis, Parkinson disease, major depression, bipolar disorder and schizophrenia. miRNA target enrichment analysis of the selected 12 miRNAs indicates an involvement of miRNAs in nervous system development, neuron projection, neuron projection development and neuron projection morphogenesis. Using this 12-miRNA signature, we differentiate between AD and controls with an accuracy of 93%, a specificity of 95% and a sensitivity of 92%. The differentiation of AD from other neurological diseases is possible with accuracies between 74% and 78%. The differentiation of the other CNS disorders from controls yields even higher accuracies. CONCLUSIONS: The data indicate that deregulated miRNAs in blood might be used as biomarkers in the diagnosis of AD or other neurological diseases.

Next-generation sequencing identifies novel microRNAs in peripheral blood of lung cancer patients.

MicroRNAs (miRNAs) are increasingly envisaged as biomarkers for various tumor and non-tumor diseases. MiRNA biomarker identification is, as of now, mostly performed in a candidate approach, limiting discovery to annotated miRNAs and ignoring unknown ones with potential diagnostic value. Here, we applied high-throughput SOLiD transcriptome sequencing of miRNAs expressed in human peripheral blood of patients with lung cancer. We developed a bioinformatics pipeline to generate profiles of miRNA markers and to detect novel miRNAs with diagnostic information. Applying our approach, we detected 76 previously unknown miRNAs and 41 novel mature forms of known precursors. In addition, we identified 32 annotated and seven unknown miRNAs that were significantly altered in cancer patients. These results demonstrate that deep sequencing of small RNAs bears high potential to quantify miRNAs in peripheral blood and to identify previously unknown miRNAs serving as biomarker for lung cancer.

A flexible and fully integrated system for amplification, detection and genotyping of genomic DNA targets based on microfluidic oligonucleotide arrays.

A strategy allowing for amplification, detection and genotyping of different genomic DNA targets in a single reaction container is described. The method makes use of primer-directed solution-phase amplification with integrated labeling in a closed, microfluidic oligonucleotide array. Selective array probes allow for subsequent detection and genotyping of generated amplicons by hybridization. The array contains up to 15,624 programmable features that can be designed, de novo synthesized and tested within 24 hours using an automated benchtop microarray synthesizer. This enables rapid prototyping and adaptation of the system to newly emerging targets such as pathogenic bacterial or viral subtypes. The system was evaluated by amplifying and detecting different loci of viral (HPV), bacterial (Bacillus sp.) and eukaryotic (human) genomes. Multiplex PCR and semi-quantitative detection with excellent detection limits of <100 target copies is hereby demonstrated. The high automation grade of the system reduces contamination risk and workload and should enhance safety and reproducibility.

High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing.

The construction of synthetic biological systems involving millions of nucleotides is limited by the lack of high-quality synthetic DNA. Consequently, the field requires advances in the accuracy and scale of chemical DNA synthesis and in the processing of longer DNA assembled from short fragments. Here we describe a highly parallel and miniaturized method, called megacloning, for obtaining high-quality DNA by using next-generation sequencing (NGS) technology as a preparative tool. We demonstrate our method by processing both chemically synthesized and microarray-derived DNA oligonucleotides with a robotic system for imaging and picking beads directly off of a high-throughput pyrosequencing platform. The method can reduce error rates by a factor of 500 compared to the starting oligonucleotide pool generated by microarray. We use DNA obtained by megacloning to assemble synthetic genes. In principle, millions of DNA fragments can be sequenced, characterized and sorted in a single megacloner run, enabling constructive biology up to the megabase scale.