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Systematic comparison of small RNA library preparation protocols for next-generation sequencing
Cloelia Dard-Dascot1, Delphine Naquin1, Yves d'Aubenton-Carafa1
1Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud, Université Paris-Saclay, 9198, Gif sur Yvette Cedex, France.
BMC Genomics
|February 7, 2018
Summary
Bias in small RNA (sRNA) library preparation can be reduced. Modified TruSeq and NEXTflex protocols using randomized adapters and PEG improve detection of 2'-O-methyl RNAs.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Next-generation sequencing revolutionized small RNA (sRNA) studies.
- Classical library preparation methods introduce bias, especially during adapter ligation.
- 3'-terminal 2'-O-methyl (2'-OMe) modification on sRNAs inhibits ligation, complicating library prep.
Purpose of the Study:
- Compare bias levels in various sRNA library preparation kits.
- Evaluate detection of normal and 2'-OMe modified RNAs.
- Identify improved conditions for sRNA library preparation.
Main Methods:
- Tested NEBNext, NEXTflex V2, SMARTer, CATS, and Illumina TruSeq kits.
- Assessed performance with standard protocols and modified versions.
- Incorporated polyethylene glycol (PEG) and randomized adapters (MRL).
Main Results:
- Ligation-free SMARTer and CATS kits showed low bias but poor performance with biological samples.
- NEXTflex kit detected the highest number of unique microRNAs (miRNAs).
- Modified TruSeq and NEXTflex protocols with randomized adapters and PEG enhanced 2'-OMe RNA detection.
Conclusions:
- Ligation-free protocols were not superior despite common assumptions about ligation bias.
- Modified TruSeq and NEXTflex protocols offer improved tools for studying 2'-OMe RNAs.
- Optimized library preparation is crucial for accurate sRNA profiling.
