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10.1186/1471-2164-15-401 http://scihub22266oqcxt.onion/10.1186/1471-2164-15-401 C4045971!4045971 !24886553     free     free     free Warning: file_get_contents(https://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=24886553 &cmd=llinks): Failed to open stream: HTTP request failed! 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Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage |pdf=|usr=}}{{24886553 }} gab.com Text Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage JO: BMC Genomics http://www.kidney.de/pget.php?&tw=1&pmid=24886553 #FOAvip BACKGROUND: A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). RESULTS: Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. CONCLUSION: We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms. Twit Text FOAVip Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage ????BMC Genomics http://www.kidney.de/pget.php?&tw=1&pmid=24886553 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=24886553 #FOAvip English Wikipedia <ref>{{Cite pmid|24886553 }}</ref> Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage #MMPMID24886553 Archer SK ; Shirokikh NE ; Preiss T BMC Genomics 2014[May]; 15 (1 ): 401 PMID24886553 show ga BACKGROUND: A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). RESULTS: Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. CONCLUSION: We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms. |DNA Probes/metabolism [MESH] |DNA, Complementary/*metabolism [MESH] |Gene Library [MESH] |High-Throughput Nucleotide Sequencing [MESH] |RNA/chemistry/metabolism [MESH] |Saccharomyces cerevisiae/genetics [MESH]Selective and flexible depletion of problematic sequences from RNA-seq(epmc).pdf DeepDyve Pubget Overpricing