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1.
PLoS One ; 10(5): e0126911, 2015.
Article in English | MEDLINE | ID: mdl-25965996

ABSTRACT

Genetic variation modulates gene expression transcriptionally or post-transcriptionally, and can profoundly alter an individual's phenotype. Measuring allelic differential expression at heterozygous loci within an individual, a phenomenon called allele-specific expression (ASE), can assist in identifying such factors. Massively parallel DNA and RNA sequencing and advances in bioinformatic methodologies provide an outstanding opportunity to measure ASE genome-wide. In this study, matched DNA and RNA sequencing, genotyping arrays and computationally phased haplotypes were integrated to comprehensively and conservatively quantify ASE in a single human brain and liver tissue sample. We describe a methodological evaluation and assessment of common bioinformatic steps for ASE quantification, and recommend a robust approach to accurately measure SNP, gene and isoform ASE through the use of personalized haplotype genome alignment, strict alignment quality control and intragenic SNP aggregation. Our results indicate that accurate ASE quantification requires careful bioinformatic analyses and is adversely affected by sample specific alignment confounders and random sampling even at moderate sequence depths. We identified multiple known and several novel ASE genes in liver, including WDR72, DSP and UBD, as well as genes that contained ASE SNPs with imbalance direction discordant with haplotype phase, explainable by annotated transcript structure, suggesting isoform derived ASE. The methods evaluated in this study will be of use to researchers performing highly conservative quantification of ASE, and the genes and isoforms identified as ASE of interest to researchers studying those loci.


Subject(s)
DNA/analysis , Gene Expression Profiling/methods , RNA/analysis , Sequence Analysis, RNA/methods , Allelic Imbalance , Brain/metabolism , Humans , Liver/metabolism , Polymorphism, Single Nucleotide , RNA Isoforms , Sequence Analysis, DNA/methods
2.
Biotechniques ; 57(1): 31-8, 2014 Jul.
Article in English | MEDLINE | ID: mdl-25005691

ABSTRACT

Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.


Subject(s)
Chromosome Breakpoints , Chromosomes, Human , High-Throughput Nucleotide Sequencing/methods , Base Sequence , Carcinoma, Pancreatic Ductal/genetics , Chromosome Aberrations , DNA Primers , Humans , Molecular Sequence Data , Neoplasms/genetics , Pancreatic Neoplasms/genetics , Polymerase Chain Reaction/methods , Workflow
3.
Genome Biol ; 15(3): R51, 2014 Mar 14.
Article in English | MEDLINE | ID: mdl-24629056

ABSTRACT

BACKGROUND: MicroRNAs (miRNAs) bind to mRNAs and target them for translational inhibition or transcriptional degradation. It is thought that most miRNA-mRNA interactions involve the seed region at the 5' end of the miRNA. The importance of seed sites is supported by experimental evidence, although there is growing interest in interactions mediated by the central region of the miRNA, termed centered sites. To investigate the prevalence of these interactions, we apply a biotin pull-down method to determine the direct targets of ten human miRNAs, including four isomiRs that share centered sites, but not seeds, with their canonical partner miRNAs. RESULTS: We confirm that miRNAs and their isomiRs can interact with hundreds of mRNAs, and that imperfect centered sites are common mediators of miRNA-mRNA interactions. We experimentally demonstrate that these sites can repress mRNA activity, typically through translational repression, and are enriched in regions of the transcriptome bound by AGO. Finally, we show that the identification of imperfect centered sites is unlikely to be an artifact of our protocol caused by the biotinylation of the miRNA. However, the fact that there was a slight bias against seed sites in our protocol may have inflated the apparent prevalence of centered site-mediated interactions. CONCLUSIONS: Our results suggest that centered site-mediated interactions are much more frequent than previously thought. This may explain the evolutionary conservation of the central region of miRNAs, and has significant implications for decoding miRNA-regulated genetic networks, and for predicting the functional effect of variants that do not alter protein sequence.


Subject(s)
MicroRNAs/metabolism , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Binding Sites , Genome, Human , HEK293 Cells , Humans , MicroRNAs/genetics , RNA, Messenger/genetics
4.
PLoS One ; 8(11): e74380, 2013.
Article in English | MEDLINE | ID: mdl-24250782

ABSTRACT

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.


Subject(s)
Computational Biology , Neoplasms/genetics , Point Mutation/genetics , Software , DNA Copy Number Variations/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Mutation , Neoplasms/pathology , Polymerase Chain Reaction/methods
5.
RNA ; 19(12): 1767-80, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24158791

ABSTRACT

Metastasis is a complex, multistep process involved in the progression of cancer from a localized primary tissue to distant sites, often characteristic of the more aggressive forms of this disease. Despite being studied in great detail in recent years, the mechanisms that govern this process remain poorly understood. In this study, we identify a novel role for miR-139-5p in the inhibition of breast cancer progression. We highlight its clinical relevance by reviewing miR-139-5p expression across a wide variety of breast cancer subtypes using in-house generated and online data sets to show that it is most frequently lost in invasive tumors. A biotin pull-down approach was then used to identify the mRNA targets of miR-139-5p in the breast cancer cell line MCF7. Functional enrichment analysis of the pulled-down targets showed significant enrichment of genes in pathways previously implicated in breast cancer metastasis (P < 0.05). Further bioinformatic analysis revealed a predicted disruption to the TGFß, Wnt, Rho, and MAPK/PI3K signaling cascades, implying a potential role for miR-139-5p in regulating the ability of cells to invade and migrate. To corroborate this finding, using the MDA-MB-231 breast cancer cell line, we show that overexpression of miR-139-5p results in suppression of these cellular phenotypes. Furthermore, we validate the interaction between miR-139-5p and predicted targets involved in these pathways. Collectively, these results suggest a significant functional role for miR-139-5p in breast cancer cell motility and invasion and its potential to be used as a prognostic marker for the aggressive forms of breast cancer.


Subject(s)
Carcinoma, Ductal, Breast/genetics , Gene Expression Regulation, Neoplastic , MicroRNAs/physiology , Triple Negative Breast Neoplasms/genetics , Base Sequence , Binding Sites , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Ductal, Breast/secondary , Cell Line, Tumor , Cell Movement , Cell Proliferation , DNA Replication , Female , Gene Expression , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Neoplasm Invasiveness , RNA Interference , Signal Transduction , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology
6.
RNA ; 19(2): 230-42, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23249749

ABSTRACT

MicroRNAs are noncoding regulators of gene expression, which act by repressing protein translation and/or degrading mRNA. Many have been shown to drive tumorigenesis in cancer, but functional studies to understand their mode of action are typically limited to single-target genes. In this study, we use synthetic biotinylated miRNA to pull down endogenous targets of miR-182-5p. We identified more than 1000 genes as potential targets of miR-182-5p, most of which have a known function in pathways underlying tumor biology. Specifically, functional enrichment analysis identified components of both the DNA damage response pathway and cell cycle to be highly represented in this target cohort. Experimental validation confirmed that miR-182-5p-mediated disruption of the homologous recombination (HR) pathway is a consequence of its ability to target multiple components in that pathway. Although there is a strong enrichment for the cell cycle ontology, we do not see primary proliferative defects as a consequence of miR-182-5p overexpression. We highlight targets that could be responsible for miR-182-5p-mediated disruption of other biological processes attributed in the literature so far. Finally, we show that miR-182-5p is highly expressed in a panel of human breast cancer samples, highlighting its role as a potential oncomir in breast cancer.


Subject(s)
BRCA1 Protein/metabolism , Breast Neoplasms/genetics , DNA Repair/genetics , MicroRNAs/metabolism , BRCA1 Protein/genetics , Breast Neoplasms/metabolism , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation , Cluster Analysis , Cohort Studies , DNA Damage , Female , Gene Expression Profiling , HeLa Cells , Homologous Recombination/genetics , Humans , MicroRNAs/genetics , Models, Genetic , Oligonucleotide Array Sequence Analysis , Up-Regulation
7.
Genome Biol ; 12(12): R126, 2011 Dec 30.
Article in English | MEDLINE | ID: mdl-22208850

ABSTRACT

BACKGROUND: Variants of microRNAs (miRNAs), called isomiRs, are commonly reported in deep-sequencing studies; however, the functional significance of these variants remains controversial. Observational studies show that isomiR patterns are non-random, hinting that these molecules could be regulated and therefore functional, although no conclusive biological role has been demonstrated for these molecules. RESULTS: To assess the biological relevance of isomiRs, we have performed ultra-deep miRNA-seq on ten adult human tissues, and created an analysis pipeline called miRNA-MATE to align, annotate, and analyze miRNAs and their isomiRs. We find that isomiRs share sequence and expression characteristics with canonical miRNAs, and are generally strongly correlated with canonical miRNA expression. A large proportion of isomiRs potentially derive from AGO2 cleavage independent of Dicer. We isolated polyribosome-associated mRNA, captured the mRNA-bound miRNAs, and found that isomiRs and canonical miRNAs are equally associated with translational machinery. Finally, we transfected cells with biotinylated RNA duplexes encoding isomiRs or their canonical counterparts and directly assayed their mRNA targets. These studies allow us to experimentally determine genome-wide mRNA targets, and these experiments showed substantial overlap in functional mRNA networks suppressed by both canonical miRNAs and their isomiRs. CONCLUSIONS: Together, these results find isomiRs to be biologically relevant and functionally cooperative partners of canonical miRNAs that act coordinately to target pathways of functionally related genes. This work exposes the complexity of the miRNA-transcriptome, and helps explain a major miRNA paradox: how specific regulation of biological processes can occur when the specificity of miRNA targeting is mediated by only 6 to 11 nucleotides.


Subject(s)
Argonaute Proteins/genetics , Gene Regulatory Networks/genetics , MicroRNAs/genetics , RNA, Messenger/genetics , Base Sequence , Biotinylation , DEAD-box RNA Helicases/genetics , Gene Expression Profiling , HEK293 Cells , HeLa Cells , High-Throughput Nucleotide Sequencing , Humans , MicroRNAs/classification , MicroRNAs/isolation & purification , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Ribonuclease III/genetics , Sequence Alignment , Transcriptome , Transfection
8.
Nat Genet ; 41(5): 563-71, 2009 May.
Article in English | MEDLINE | ID: mdl-19377475

ABSTRACT

Although repetitive elements pervade mammalian genomes, their overall contribution to transcriptional activity is poorly defined. Here, as part of the FANTOM4 project, we report that 6-30% of cap-selected mouse and human RNA transcripts initiate within repetitive elements. Analysis of approximately 250,000 retrotransposon-derived transcription start sites shows that the associated transcripts are generally tissue specific, coincide with gene-dense regions and form pronounced clusters when aligned to full-length retrotransposon sequences. Retrotransposons located immediately 5' of protein-coding loci frequently function as alternative promoters and/or express noncoding RNAs. More than a quarter of RefSeqs possess a retrotransposon in their 3' UTR, with strong evidence for the reduced expression of these transcripts relative to retrotransposon-free transcripts. Finally, a genome-wide screen identifies 23,000 candidate regulatory regions derived from retrotransposons, in addition to more than 2,000 examples of bidirectional transcription. We conclude that retrotransposon transcription has a key influence upon the transcriptional output of the mammalian genome.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation , Retroelements/genetics , 3' Untranslated Regions/genetics , 3' Untranslated Regions/metabolism , Animals , Cells, Cultured , Humans , Mammals/genetics , Mice , Promoter Regions, Genetic , RNA, Messenger , RNA, Untranslated/metabolism
9.
Genome Biol ; 9(8): R127, 2008.
Article in English | MEDLINE | ID: mdl-18700987

ABSTRACT

BACKGROUND: MicroRNAs are modifiers of gene expression, acting to reduce translation through either translational repression or mRNA cleavage. Recently, it has been shown that some microRNAs can act to promote or suppress cell transformation, with miR-17-92 described as the first oncogenic microRNA. The association of miR-17-92 encoded microRNAs with a surprisingly broad range of cancers not only underlines the clinical significance of this locus, but also suggests that miR-17-92 may regulate fundamental biological processes, and for these reasons miR-17-92 has been considered as a therapeutic target. RESULTS: In this study, we show that miR-17-92 is a cell cycle regulated locus, and ectopic expression of a single microRNA (miR-17-5p) is sufficient to drive a proliferative signal in HEK293T cells. For the first time, we reveal the mechanism behind this response - miR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes involved in the transition between these phases. While both pro- and anti-proliferative genes are targeted by miR-17-5p, pro-proliferative mRNAs are specifically up-regulated by secondary and/or tertiary effects in HEK293T cells. CONCLUSION: The miR-17-5p microRNA is able to act as both an oncogene and a tumor suppressor in different cellular contexts; our model of competing positive and negative signals can explain both of these activities. The coordinated suppression of proliferation-inhibitors allows miR-17-5p to efficiently de-couple negative regulators of the MAPK (mitogen activated protein kinase) signaling cascade, promoting growth in HEK293T cells. Additionally, we have demonstrated the utility of a systems biology approach as a unique and rapid approach to uncover microRNA function.


Subject(s)
G1 Phase/physiology , MicroRNAs/physiology , S Phase/physiology , Cell Line , Cell Proliferation , Humans , Mitogen-Activated Protein Kinase 9/genetics , Mitogen-Activated Protein Kinase 9/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , Signal Transduction
10.
Nat Methods ; 5(7): 613-9, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18516046

ABSTRACT

We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)(+) transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.


Subject(s)
Embryonic Stem Cells/metabolism , Gene Expression Profiling/methods , RNA, Messenger/genetics , Sequence Analysis, RNA/methods , Animals , Cell Differentiation , Embryonic Stem Cells/cytology , Expressed Sequence Tags , Gene Expression Profiling/statistics & numerical data , Gene Library , Mice , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Polymorphism, Single Nucleotide , Sensitivity and Specificity , Signal Transduction
11.
Differentiation ; 75(5): 337-49, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17286599

ABSTRACT

Embryonic stem (ES) cells have the capacity to differentiate into all cells of the developing embryo and may provide a renewable resource for future cell replacement therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ES cell culture has previously been shown to induce transcription factors, signaling molecules, and cell adhesion proteins expressed during mesoderm specification of the embryo. Here, we show the dynamics of primitive streak mesoderm differentiation in ES cells is comparable between serum and serum-free embryoid body (EB) cultures, supplemented with BMP4. Furthermore, we show a delayed wave of expression of a cohort of genes (Pax2, WT1, podocalyxin, pod-1, and nephrin), which play important roles during embryonic kidney development. The paired box transcription factor, Pax2, is one of the earliest genes expressed during kidney organogenesis and is required for normal urogenital development. ES cell lines containing either a modified Pax2 promoter-lacZ or bacterial artificial chromosome-green fluorescent protein (GFP) transgene were generated, which enabled the quantitative analysis of kidney rather than neuronal Pax2 expression within EBs. Both beta-galactosidase activity and GFP expression were detected by immunohistochemical and flow cytometric analysis following 16 days of EB culture, which correlated with an increase in Pax2 transcript levels. Together, these results suggest a spontaneous kidney gene expression program develops in mature EBs grown in both serum and serum-free conditions, when supplemented with BMP4. Further, the recombinant growth factors BMP2, BMP4, and BMP7 strongly influence gene expression within mesoderm induced EBs. BMP4 promotes ventral (blood) and intermediate (kidney) mesoderm gene expression, whereas BMP2 and BMP7 promote kidney outcomes at the expense of hematopoietic commitment. This induction assay and these unique ES cell lines will be useful for the generation of mesoderm-derived cell populations with implications for future cell therapeutic/integration assays.


Subject(s)
Cell Differentiation , Cell Lineage , Embryonic Stem Cells/cytology , Kidney/cytology , Animals , Bone Morphogenetic Protein 2 , Bone Morphogenetic Protein 4 , Bone Morphogenetic Protein 7 , Bone Morphogenetic Proteins/metabolism , Cell Proliferation , Chromosomes, Artificial, Bacterial/genetics , Culture Media, Serum-Free , Embryonic Stem Cells/metabolism , Flow Cytometry , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hematopoiesis , Immunoenzyme Techniques , Kidney/metabolism , Mesoderm/metabolism , Mice , PAX2 Transcription Factor/metabolism , Transforming Growth Factor beta/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
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