Dual randomization of oligonucleotides to reduce the bias in ribosome-profiling libraries.
Methods
; 107: 89-97, 2016 09 01.
Article
en En
| MEDLINE
| ID: mdl-27450428
ABSTRACT
Protein translation is at the heart of cellular metabolism and its in-depth characterization is key for many lines of research. Recently, ribosome profiling became the state-of-the-art method to quantitatively characterize translation dynamics at a transcriptome-wide level. However, the strategy of library generation affects its outcomes. Here, we present a modified ribosome-profiling protocol starting from yeast, human cells and vertebrate brain tissue. We use a DNA linker carrying four randomized positions at its 5' end and a reverse-transcription (RT) primer with three randomized positions to reduce artifacts during library preparation. The use of seven randomized nucleotides allows to efficiently detect library-generation artifacts. We find that the effect of polymerase chain reaction (PCR) artifacts is relatively small for global analyses when sufficient input material is used. However, when input material is limiting, our strategy improves the sensitivity of gene-specific analyses. Furthermore, randomized nucleotides alleviate the skewed frequency of specific sequences at the 3' end of ribosome-protected fragments (RPFs) likely resulting from ligase specificity. Finally, strategies that rely on dual ligation show a high degree of gene-coverage variation. Taken together, our approach helps to remedy two of the main problems associated with ribosome-profiling data. This will facilitate the analysis of translational dynamics and increase our understanding of the influence of RNA modifications on translation.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Ribosomas
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Ingeniería Genética
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Perfilación de la Expresión Génica
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Secuenciación de Nucleótidos de Alto Rendimiento
Tipo de estudio:
Clinical_trials
Límite:
Humans
Idioma:
En
Año:
2016
Tipo del documento:
Article