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An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments.

ABSTRACT: Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2-3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.

SUBMITTER: Heyer EE

PROVIDER: S-EPMC4288154 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments.

Heyer Erin E EE Ozadam Hakan H Ricci Emiliano P EP Cenik Can C Moore Melissa J MJ

Nucleic acids research 20141212 1

Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting ...[more]

PMID: 25505164

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An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments

Project description:Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs, variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2–3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.

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An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments

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Genome-scale validation of deep-sequencing libraries.

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Strand-specific deep sequencing of the transcriptome.

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Error analysis of deep sequencing of phage libraries: peptides censored in sequencing.

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Project description:We report an alternative approach to transcriptome sequencing for the Illumina Genome Analyzer, in which the reverse transcription reaction takes place on the flowcell. No amplification is performed during the library preparation, so PCR biases and duplicates are avoided, and because the template is poly(A)(+) RNA rather than cDNA, the resulting sequences are necessarily strand-specific. The method is compatible with paired- or single-end sequencing.

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An optimized ribodepletion approach for C. elegans RNA-sequencing libraries

Project description:Advances over the past decade have allowed for increasingly fine-grained labeling and isolation of rare cell samples for transcriptomic analysis, providing new insights into cell function and gene regulation. These samples often contain very little RNA for sequencing, and so have required new techniques to capture and amplify transcripts of interest. However, as new tools are developed, they are often optimized for mammalian samples. Thus, it is unclear for invertebrate samples what the best practices are for library preparation, and how differences in library preparation approaches affect final results. Here we compared two commercially available techniques: a commonly used polyA selection approach, and a newly developed ribodepletion approach, for which we designed a unique C. elegans-specific probe set. We performed a detailed comparison on multiple RNA samples and developed novel analysis methods to compare the results. Our analysis identifies clear strengths and weaknesses for both of these approaches in building libraries from low abundance RNA samples in C. elegans. We find that the polyA approach is superior in both percent reads mapped and rRNA removal, while the ribodepletion approach provides better detection for long and noncoding RNAs along with increased certainty of calling expression for low abundance transcripts. These insights will help guide researchers in designing future experiments in transcriptomics and provide a model for comparative analysis of library-building techniques.

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Targeted Deep Sequencing Uncovers Cryptic KIT Mutations in KIT/PDGFRA/SDH/RAS-P Wild-Type GIST.

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