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Small RNA sequencing of plasma RNA

ABSTRACT: We report the sequencing of small RNAs present in the plasma of three normal subjects. In addition to microRNAs we identified abundant non-human small RNA sequences. The organisms from which these were derived were identified by BLAST searches with contigs assembled from the short sequences. The taxonomic profiles were very consistent between individuals, including plants and microbes reported previously in the microbiome, but in proportions distinct from other sites. The majority of bacterial reads were from the phylum Proteobacteria, whilst for 5 of 6 individuals over 90% of the more abundant fungal reads were from the phylum Ascomycota; of these over 90% were from the order Hypocreales. Most reads mapped to rRNA sequences and the presence of specific common sequences was confirmed by RT-PCR. In addition, extremely abundant small RNAs derived from human Y RNAs were detected. We conclude that a characteristic profile of a subset of the human microbiome can be obtained by sequencing small RNAs present in the blood. The origin and potential function of these molecules remains to be determined, but the specific profile is likely to reflect health status. This facile test has immense potential to provide biomarkers for the diagnosis and prognosis of human disease. The profile of small RNAs present in the plasma of three normal subjects was determined

ORGANISM(S): Homo sapiens

SUBMITTER: David Simpson

PROVIDER: E-GEOD-52981 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Small RNA sequencing of plasma RNA

Project description:We report the sequencing of small RNAs present in the plasma of three normal subjects. In addition to microRNAs we identified abundant non-human small RNA sequences. The organisms from which these were derived were identified by BLAST searches with contigs assembled from the short sequences. The taxonomic profiles were very consistent between individuals, including plants and microbes reported previously in the microbiome, but in proportions distinct from other sites. The majority of bacterial reads were from the phylum Proteobacteria, whilst for 5 of 6 individuals over 90% of the more abundant fungal reads were from the phylum Ascomycota; of these over 90% were from the order Hypocreales. Most reads mapped to rRNA sequences and the presence of specific common sequences was confirmed by RT-PCR. In addition, extremely abundant small RNAs derived from human Y RNAs were detected. We conclude that a characteristic profile of a subset of the human microbiome can be obtained by sequencing small RNAs present in the blood. The origin and potential function of these molecules remains to be determined, but the specific profile is likely to reflect health status. This facile test has immense potential to provide biomarkers for the diagnosis and prognosis of human disease.

2014-09-29 | GSE52981 | GEO

High-throughput sequencing analysis identify miRNAs in 7dpf F1-zebrafish under Î²-diketone antibiotics exposure to F0-zebrafish

Project description:Small RNA high-throughput sequencing technology was used to characterize the miRNAs in F1-zebrafish after 90-day Î²-diketoneÂ antibiotic (DKA) exposure to F0-zebrafish at 6.25 and 12.5 mg/L. The small RNA libraries from 7-dpf F1-zebrafish were constructed. In total, 10,117,347, 9,818,830 and 12,049,949 raw reads were acquired, respectively, under the different DKA-exposure treatments (0, 6.25 mg/L and 12.5mg/L) from the three miRNAs libraries by Illumina sequencing. Low-quality reads were removed, which included 5' contaminants, those missing the 3' primer or insert tag, sequences with a poly A tail, and those shorter than 17 nt and longer than 25 nt. As a result, 8,141,146 (representing 312,735 unique sequences; control), 8,687,210 (representing 251,508 unique sequences; 6.25 mg/L), and 10,569,566 (representing 441,938 unique sequences; 12.5 mg/L) valid reads in the 17 to 25 nt size range were isolated for further analysis. The sRNAs from the three libraries were similar, and the unique sRNA reads were mainly distributed in the 20-24 nt range, among which 22 and 23 nt accounted for 41.8% and 20.0% of total unique sRNA reads, respectively. The 22-nt sRNAs were the most abundant, with the length distribution of counts of sequ-seqs and unique miRNAs displaying a normal distribution. Sample 1: Examination of small RNA in 7-dpf F1-zebrafish after 90-day DKA exposure to F0-zebrafish at 0 mg/L; Sample 2: Examination of small RNA in 7-dpf F1-zebrafish after 90-day DKA exposure to F0-zebrafish at 6.25 mg/L; Sample 3: Examination of small RNA in 7-dpf F1-zebrafish after 90-day DKA exposure to F0-zebrafish at 12.5 mg/L.

2016-07-30 | E-GEOD-85004 | biostudies-arrayexpress

Small RNA deep sequencing of cellular subcompartments

Project description:RNA deep sequencing efforts have revealed abundant expression of small RNAs derived from small nucleolar (sno) RNAs. We employ here spatial RNA deep sequencing to assess the expression of 10-40 nt small RNAs in subcellular compartments of HeLa cells. Total cellular, cytoplasmic, nuclear and nucleolar fractions were isolated, RNA was purified and size-fractionated in a two-step process to yield 10-40 nt RNA fractions. cDNA libraries were constructed and sequenced on a Ion Torrent platform. Vast majority of cellular, cytoplasmic and nuclear small RNA reads were identified as miRNAs. We found the expression of eleven ten miRNAs in the nucleolar preparations using a cut-off rate of 10 reads. Several miRNAs had a greater relative abundance in the nucleolus compared to the other compartments. The nucleolar small RNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped both 5M-bM-^@M-^Y and 3M-bM-^@M-^Y ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads from SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as 20 and 25 nt RNAs. This study reveals a rich representation of cell-compartment specific expression of small RNAs and the distinctive unique composition of the nucleolar small RNAs. Examination of small RNAs in cellular subcompartments

2014-05-07 | E-GEOD-50057 | biostudies-arrayexpress

Total exRNA Profiles from Plasma, Saliva, and Urine of Healthy Subjects

Project description:<p>There are currently few datasets describing baseline expression levels for total cell-free circulating RNA from healthy control subjects. Total extracellular RNA was isolated from plasma, urine, and saliva samples from healthy controls. We sequenced small RNAs from 183 plasma samples, 204 urine samples and 46 saliva samples from 55 college athletes ages 18-25 years. Many of the participants provided more than one sample, weeks or months apart, allowing us to assess variability in an individual's exRNA expression levels over time. Several individuals provided all three biofluid types at one time, producing data on individual expression levels across several biofluid types. Here we provide a systematic analysis of small exRNAs present in each biofluid, as well as an analysis of exogenous RNAs. We find that a large number of RNA fragments in plasma (63%) and urine (54%) have sequences that are assigned to YRNA and tRNA fragments respectively. Surprisingly, while many miRNAs can be detected, there are few miRNAs that are consistently detected in all samples from a single biofluid.</p>

| phs001258 | dbGaP

High-throughput sequencing analysis identify miRNAs in 7dpf F1-zebrafish under β-diketone antibiotics exposure to F0-zebrafish

Project description:Small RNA high-throughput sequencing technology was used to characterize the miRNAs in F1-zebrafish after 90-day β-diketone antibiotic (DKA) exposure to F0-zebrafish at 6.25 and 12.5 mg/L. The small RNA libraries from 7-dpf F1-zebrafish were constructed. In total, 10,117,347, 9,818,830 and 12,049,949 raw reads were acquired, respectively, under the different DKA-exposure treatments (0, 6.25 mg/L and 12.5mg/L) from the three miRNAs libraries by Illumina sequencing. Low-quality reads were removed, which included 5' contaminants, those missing the 3' primer or insert tag, sequences with a poly A tail, and those shorter than 17 nt and longer than 25 nt. As a result, 8,141,146 (representing 312,735 unique sequences; control), 8,687,210 (representing 251,508 unique sequences; 6.25 mg/L), and 10,569,566 (representing 441,938 unique sequences; 12.5 mg/L) valid reads in the 17 to 25 nt size range were isolated for further analysis. The sRNAs from the three libraries were similar, and the unique sRNA reads were mainly distributed in the 20-24 nt range, among which 22 and 23 nt accounted for 41.8% and 20.0% of total unique sRNA reads, respectively. The 22-nt sRNAs were the most abundant, with the length distribution of counts of sequ-seqs and unique miRNAs displaying a normal distribution.

2016-07-30 | GSE85004 | GEO

Small RNA deep sequencing of cellular subcompartments

Project description:RNA deep sequencing efforts have revealed abundant expression of small RNAs derived from small nucleolar (sno) RNAs. We employ here spatial RNA deep sequencing to assess the expression of 10-40 nt small RNAs in subcellular compartments of HeLa cells. Total cellular, cytoplasmic, nuclear and nucleolar fractions were isolated, RNA was purified and size-fractionated in a two-step process to yield 10-40 nt RNA fractions. cDNA libraries were constructed and sequenced on a Ion Torrent platform. Vast majority of cellular, cytoplasmic and nuclear small RNA reads were identified as miRNAs. We found the expression of eleven ten miRNAs in the nucleolar preparations using a cut-off rate of 10 reads. Several miRNAs had a greater relative abundance in the nucleolus compared to the other compartments. The nucleolar small RNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped both 5’ and 3’ ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads from SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as 20 and 25 nt RNAs. This study reveals a rich representation of cell-compartment specific expression of small RNAs and the distinctive unique composition of the nucleolar small RNAs.

2014-05-07 | GSE50057 | GEO

Preliminary exploration of Oryza sativa small RNAs - Run 1 and 2

Project description:The small RNAs presented here were produced as a preliminary exploration of small RNAs in rice, and as such, various tissues and stress conditions were sampled. Small RNAs present in these samples were all mapped to the rice genome TIGR version 5. The total number of distinct mapped sequences are 12879 for Run 1 and 88508 for Run 2. The total number of sequence reads were respectively 70406 and 191682. The datasets contain Oryza sativa var Nipponbar endogenous small RNA sequences in the size range 18 to 34 nt. Plants were grown in a Conviron Environmental Chamber at high light intensity using both high pressure sodium and metal halide lamps for 10.5 hr at 28 degrees C and for 13.5 hr at 26 degrees C in the dark. RNA was extracted from rice tissues at various stages of development and under different abiotic and biotic stresses. The small RNAs presented here were all mapped to the rice genome TIGR version 5. The total number of distinct mapped sequences are 12879 for Run 1 and 88508 for Run 2. The total number of sequence reads were respectively 70406 and 191682.

2009-06-08 | GSE16248 | GEO

KSHV oral shedding and plasma viremia result in significant changes in the extracellular tumorigenic miRNA expression profile in patients exposed to malaria

Project description:Purpose: The goal of this study is to compare the small RNA expression profile in exosomes isolated from plasma samples from KSHV+/HIV- and KSHV+/HIV+ patients Methods: Total RNA was isolated from plasma exosomes obtained from Ugandan patients with different viral status. A small RNA fraction was gel purified and was used to make sequencing libraries. At least 14 million read sequences were obtained from each library. Non-redondant sequences were then aligned to genomic (hg19) and and mRNA sequence (hg19) using bowtie 2; sequences with perfect-match and 1bp mismatch alignments were retained for further analysis using DEseq2 Methods: Total RNA was isolated from plasma obtained from KSHV-infected patients with or without HIV co-infection. A small RNA fraction was gel purified and was used to make sequencing libraries. At least 14 million read sequences were obtained from each library. Non-redondant sequences were then aligned to genomic (hg19) and and mRNA sequence (hg19) using bowtie 2; sequences with perfect-match and 1bp mismatch alignments were retained for further analysis using DEseq2 Results:Plasma exosomes isolated from either KSHV-infected or KSHV/ HIV co-infected patients exposed to malaria are overwhelmingly populated with YRNAs instead of miRNAs Results: KSHV oral sheeding correlated with the detection of a pro-tumorigenic cellualr miRNA profile in plasma. KSHV plasma viremia is associated with the detec tion of KSHV encoded miRNA in plasma Conclusions: Ugandan patients exposed to Plasmodium falciparum show an unusual repertoire of small RNAs in plasma exosomes, as the exosomal RNA is mostly composed of small RNA from the YRNA biotype Conclusions: Our study represents the first detailed analysis of the small RNA biotypes present in plasma from KSHV infected and KSHV/HIV co-infected patients without clinical sign of KSHV associated malignancies.

2018-02-09 | GSE106238 | GEO

Phospho-small RNA-seq reveals circulating, extracellular mRNA/lncRNAs as potential biomarkers in human plasma: Healthy Cohort [HEALTHYPLASMA]

Project description:Extracellular RNAs (exRNAs) in blood and other biofluids have attracted great interest as potential biomarkers in liquid biopsy applications, as well as for their potential biological functions. Whereas it is well-established that extracellular microRNAs are present in human blood circulation, the degree to which messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are represented in plasma is less clear. Here we report that mRNA and lncRNA species are present as small fragments in plasma that are not detected by standard small RNA-seq methods, because they lack 5’-phosphorylation or carry 3’-phosphorylation. We developed a modified sequencing protocol (termed “phospho-sRNA-seq”) that incorporates upfront RNA treatment with T4 polynucleotide kinase (which also has 3’ phosphatase activity) and compared it to a standard small RNA-seq protocol, using as input both a pool of synthetic RNAs with diverse 5’ and 3’ end chemistries, as well exRNA isolated from human blood plasma. Using a custom, high-stringency pipeline for data analysis we identified mRNA and lncRNA transcriptome fingerprints in plasma, including multiple tissue-specific gene sets. In this cohort of healthy individuals, we demonstrate the improved capture of mRNA and lncRNA fragments using the phospho-sRNA-seq protocol. We find they primarily arise from mRNA exons, and that abundant fragments are consistently detected in multiple individuals.

2019-05-03 | GSE126049 | GEO

Small RNA identification in Arabidopsis thaliana using modified MPSS

Project description:Small RNAs play important regulatory roles in most eukaryotes but only a small proportion of these molecules have been identified. We sequenced more than two million small RNAs from seedlings and the inflorescence of the model plant Arabidopsis thaliana. Known and new miRNAs were among the most abundant of the non-redundant set of more than 75,000 sequences, whereas more than half represented lower abundance small-interfering RNAs (siRNAs) that match repetitive sequences, intergenic regions, and genes. Individual or clusters of highly-regulated small RNAs were readily observed. Targets of antisense RNA or miRNA did not appear to be preferentially associated with siRNAs. Many genomic regions previously considered featureless were found to be sites of numerous small RNAs. Small RNAs identified in seedlings and inflorescence tissues.

2005-09-02 | E-GEOD-3008 | biostudies-arrayexpress

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