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  • 1
    Publication Date: 2008-07-05
    Description: The functional complexity of the human transcriptome is not yet fully elucidated. We report a high-throughput sequence of the human transcriptome from a human embryonic kidney and a B cell line. We used shotgun sequencing of transcripts to generate randomly distributed reads. Of these, 50% mapped to unique genomic locations, of which 80% corresponded to known exons. We found that 66% of the polyadenylated transcriptome mapped to known genes and 34% to nonannotated genomic regions. On the basis of known transcripts, RNA-Seq can detect 25% more genes than can microarrays. A global survey of messenger RNA splicing events identified 94,241 splice junctions (4096 of which were previously unidentified) and showed that exon skipping is the most prevalent form of alternative splicing.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sultan, Marc -- Schulz, Marcel H -- Richard, Hugues -- Magen, Alon -- Klingenhoff, Andreas -- Scherf, Matthias -- Seifert, Martin -- Borodina, Tatjana -- Soldatov, Aleksey -- Parkhomchuk, Dmitri -- Schmidt, Dominic -- O'Keeffe, Sean -- Haas, Stefan -- Vingron, Martin -- Lehrach, Hans -- Yaspo, Marie-Laure -- New York, N.Y. -- Science. 2008 Aug 15;321(5891):956-60. doi: 10.1126/science.1160342. Epub 2008 Jul 3.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18599741" target="_blank"〉PubMed〈/a〉
    Keywords: *Alternative Splicing ; Cell Line ; Cell Line, Tumor ; Computational Biology ; DNA, Complementary ; DNA, Intergenic ; Exons ; *Gene Expression Profiling ; *Genome, Human ; Humans ; Introns ; Oligonucleotide Array Sequence Analysis ; RNA Polymerase II/metabolism ; *RNA Splice Sites ; RNA, Messenger/*genetics ; *Sequence Analysis, RNA
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 2
    Publication Date: 2013-08-28
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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  • 3
    Publication Date: 2016-07-09
    Description: Across kingdoms, RNA interference (RNAi) has been shown to control gene expression at the transcriptional- or the post-transcriptional level. Here, we describe a mechanism which involves both aspects: truncated transgenes, which fail to produce intact mRNA, induce siRNA accumulation and silencing of homologous loci in trans in the ciliate Paramecium . We show that silencing is achieved by co-transcriptional silencing, associated with repressive histone marks at the endogenous gene. This is accompanied by secondary siRNA accumulation, strictly limited to the open reading frame of the remote locus. Our data shows that in this mechanism, heterochromatic marks depend on a variety of RNAi components. These include RDR3 and PTIWI14 as well as a second set of components, which are also involved in post-transcriptional silencing: RDR2, PTIWI13, DCR1 and CID2. Our data indicates differential processing of nascent un-spliced and long, spliced transcripts thus suggesting a hitherto-unrecognized functional interaction between post-transcriptional and co-transcriptional RNAi. Both sets of RNAi components are required for efficient trans -acting RNAi at the chromatin level and our data indicates similar mechanisms contributing to genome wide regulation of gene expression by epigenetic mechanisms.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 4
    Publication Date: 2013-09-25
    Description: The regulation of gene expression in cells, including by microRNAs (miRNAs), is a dynamic process. Current methods for identifying miRNA targets by combining sequence and miRNA and mRNA expression data do not adequately use the temporal information and thus miss important miRNAs and their targets. We developed the MIRna Dynamic...
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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  • 5
    Publication Date: 2016-06-01
    Description: Motivation: De novo transcriptome assembly is an integral part for many RNA-seq workflows. Common applications include sequencing of non-model organisms, cancer or meta transcriptomes. Most de novo transcriptome assemblers use the de Bruijn graph (DBG) as the underlying data structure. The quality of the assemblies produced by such assemblers is highly influenced by the exact word length k . As such no single k mer value leads to optimal results. Instead, DBGs over different k mer values are built and the assemblies are merged to improve sensitivity. However, no studies have investigated thoroughly the problem of automatically learning at which k mer value to stop the assembly. Instead a suboptimal selection of k mer values is often used in practice. Results: Here we investigate the contribution of a single k mer value in a multi- k mer based assembly approach. We find that a comparative clustering of related assemblies can be used to estimate the importance of an additional k mer assembly. Using a model fit based algorithm we predict the k mer value at which no further assemblies are necessary. Our approach is tested with different de novo assemblers for datasets with different coverage values and read lengths. Further, we suggest a simple post processing step that significantly improves the quality of multi- k mer assemblies. Conclusion: We provide an automatic method for limiting the number of k mer values without a significant loss in assembly quality but with savings in assembly time. This is a step forward to making multi- k mer methods more reliable and easier to use. Availability and Implementation :A general implementation of our approach can be found under: https://github.com/SchulzLab/KREATION . Supplementary information: Supplementary data are available at Bioinformatics online. Contact: mschulz@mmci.uni-saarland.de
    Print ISSN: 1367-4803
    Electronic ISSN: 1460-2059
    Topics: Biology , Computer Science , Medicine
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  • 6
    Publication Date: 2012-04-08
    Description: Motivation: High-throughput sequencing has made the analysis of new model organisms more affordable. Although assembling a new genome can still be costly and difficult, it is possible to use RNA-seq to sequence mRNA. In the absence of a known genome, it is necessary to assemble these sequences de novo , taking into account possible alternative isoforms and the dynamic range of expression values. Results: We present a software package named Oases designed to heuristically assemble RNA-seq reads in the absence of a reference genome, across a broad spectrum of expression values and in presence of alternative isoforms. It achieves this by using an array of hash lengths, a dynamic filtering of noise, a robust resolution of alternative splicing events and the efficient merging of multiple assemblies. It was tested on human and mouse RNA-seq data and is shown to improve significantly on the transABySS and Trinity de novo transcriptome assemblers. Availability and implementation: Oases is freely available under the GPL license at www.ebi.ac.uk/~zerbino/oases/ Contact: dzerbino@ucsc.edu Supplementary information: Supplementary data are available at Bioinformatics online.
    Print ISSN: 1367-4803
    Electronic ISSN: 1460-2059
    Topics: Biology , Computer Science , Medicine
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  • 7
    Publication Date: 2012-03-01
    Description: Motivation: The reliable detection of genomic variation in resequencing data is still a major challenge, especially for variants larger than a few base pairs. Sequencing reads crossing boundaries of structural variation carry the potential for their identification, but are difficult to map. Results: Here we present a method for ‘split’ read mapping, where prefix and suffix match of a read may be interrupted by a longer gap in the read-to-reference alignment. We use this method to accurately detect medium-sized insertions and long deletions with precise breakpoints in genomic resequencing data. Compared with alternative split mapping methods, SplazerS significantly improves sensitivity for detecting large indel events, especially in variant-rich regions. Our method is robust in the presence of sequencing errors as well as alignment errors due to genomic mutations/divergence, and can be used on reads of variable lengths. Our analysis shows that SplazerS is a versatile tool applicable to unanchored or single-end as well as anchored paired-end reads. In addition, application of SplazerS to targeted resequencing data led to the interesting discovery of a complete, possibly functional gene retrocopy variant. Availability: SplazerS is available from http://www.seqan.de/projects/ splazers. Contact: emde@inf.fu-berlin.de Supplementary information: Supplementary data are available at Bioinformatics online.
    Print ISSN: 1367-4803
    Electronic ISSN: 1460-2059
    Topics: Biology , Computer Science , Medicine
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  • 8
    Publication Date: 2012-03-01
    Description: Motivation: The identity of cells and tissues is to a large degree governed by transcriptional regulation. A major part is accomplished by the combinatorial binding of transcription factors at regulatory sequences, such as enhancers. Even though binding of transcription factors is sequence-specific, estimating the sequence similarity of two functionally similar enhancers is very difficult. However, a similarity measure for regulatory sequences is crucial to detect and understand functional similarities between two enhancers and will facilitate large-scale analyses like clustering, prediction and classification of genome-wide datasets. Results: We present the standardized alignment-free sequence similarity measure N 2, a flexible framework that is defined for word neighbourhoods. We explore the usefulness of adding reverse complement words as well as words including mismatches into the neighbourhood. On simulated enhancer sequences as well as functional enhancers in mouse development, N 2 is shown to outperform previous alignment-free measures. N 2 is flexible, faster than competing methods and less susceptible to single sequence noise and the occurrence of repetitive sequences. Experiments on the mouse enhancers reveal that enhancers active in different tissues can be separated by pairwise comparison using N 2. Conclusion: N 2 represents an improvement over previous alignment-free similarity measures without compromising speed, which makes it a good candidate for large-scale sequence comparison of regulatory sequences. Availability: The software is part of the open-source C++ library SeqAn ( www.seqan.de ) and a compiled version can be downloaded at http://www.seqan.de/projects/alf.html Contact: goeke@molgen.mpg.de ; vingron@molgen.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online.
    Print ISSN: 1367-4803
    Electronic ISSN: 1460-2059
    Topics: Biology , Computer Science , Medicine
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  • 9
    Publication Date: 2014-08-27
    Description: Motivation: Automatic error correction of high-throughput sequencing data can have a dramatic impact on the amount of usable base pairs and their quality. It has been shown that the performance of tasks such as de novo genome assembly and SNP calling can be dramatically improved after read error correction. While a large number of methods specialized for correcting substitution errors as found in Illumina data exist, few methods for the correction of indel errors, common to technologies like 454 or Ion Torrent, have been proposed. Results : We present Fiona, a new stand-alone read error–correction method. Fiona provides a new statistical approach for sequencing error detection and optimal error correction and estimates its parameters automatically. Fiona is able to correct substitution, insertion and deletion errors and can be applied to any sequencing technology. It uses an efficient implementation of the partial suffix array to detect read overlaps with different seed lengths in parallel. We tested Fiona on several real datasets from a variety of organisms with different read lengths and compared its performance with state-of-the-art methods. Fiona shows a constantly higher correction accuracy over a broad range of datasets from 454 and Ion Torrent sequencers, without compromise in speed. Conclusion : Fiona is an accurate parameter-free read error–correction method that can be run on inexpensive hardware and can make use of multicore parallelization whenever available. Fiona was implemented using the SeqAn library for sequence analysis and is publicly available for download at http://www.seqan.de/projects/fiona . Contact : mschulz@mmci.uni-saarland.de or hugues.richard@upmc.fr Supplementary information: Supplementary data are available at Bioinformatics online.
    Print ISSN: 1367-4803
    Electronic ISSN: 1460-2059
    Topics: Biology , Computer Science , Medicine
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  • 10
    Publication Date: 2013-05-29
    Description: Sequencing of RNAs (RNA-Seq) has revolutionized the field of transcriptomics, but the reads obtained often contain errors. Read error correction can have a large impact on our ability to accurately assemble transcripts. This is especially true for de novo transcriptome analysis, where a reference genome is not available. Current read error correction methods, developed for DNA sequence data, cannot handle the overlapping effects of non-uniform abundance, polymorphisms and alternative splicing. Here we present SEquencing Error CorrEction in Rna-seq data (SEECER), a hidden Markov Model (HMM)–based method, which is the first to successfully address these problems. SEECER efficiently learns hundreds of thousands of HMMs and uses these to correct sequencing errors. Using human RNA-Seq data, we show that SEECER greatly improves on previous methods in terms of quality of read alignment to the genome and assembly accuracy. To illustrate the usefulness of SEECER for de novo transcriptome studies, we generated new RNA-Seq data to study the development of the sea cucumber Parastichopus parvimensis . Our corrected assembled transcripts shed new light on two important stages in sea cucumber development. Comparison of the assembled transcripts to known transcripts in other species has also revealed novel transcripts that are unique to sea cucumber, some of which we have experimentally validated. Supporting website: http://sb.cs.cmu.edu/seecer/ .
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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