Advanced sequencing techniques have enabled the characterization and discovery of miRNAs to explode like an Icelandic volcano. But no matter how fancy your sequencing box is, the quality of your data still relies on the strength of your miRNA library preparation protocol.
A group of curious scientists from the Beijing Genomics Institute and the Chinese Academy of Sciences looked into three popular miRNA library prep methods (Life Tech’s SOLiD, Illumina version 1 and 1.5) to find out if there is any sequencing bias caused by the library prep process. (For more background on the topic, check out Nature Methods, Linsen et.al. 2009) The team used sequencing by synthesis (SBS), standard cloning and sequencing, and qRT-PCR to compare the methods. Here’s how the three library construction protocols stacked up:
- The SOLiD protocol showed higher bias for miRNA length distribution, sequence variation, end secondary structure (ESS) and reads mapping to unknown miRNAs. This is most likely due to SOLiDs use of 6 random nucleotides in the adapter ligation step.
- Illumina’s V 1.5 protocol had more 3’ ESS than the longer V1 method, perhaps because the T4 RNA Ligase 2 that’s used in the V1.5 allows ligation despite double-stranded structure on the 3’ end.
- SOLiD library prep showed the highest correlation with qRT-PCR results for miRNA quantitation and fold-change experiments, indicating that the hybridization-based approach shared by SOLiD and qRT-PCR may be better suited to those types of experiments.
No matter what miRNA sequencing library construction protocol you use, it seems, you’re bound to introduce some bias, but if you understand their strengths and weaknesses you’ll be able to pick the best method for your needs.
Overcome your bias by checking out the full article at BMC Biotechnology, September 2010