This webinar addresses improvements in the library prep workflow for small RNA sequencing in serum and plasma.
Sequencing of small RNAs from extracellular fluids, including serum and plasma, is a fast-growing area of biomarker research. Analysis of plasma and serum by small RNA sequencing involves isolating RNA, performing library preparation, sequencing, and downstream data analysis.
Our speaker, David Simpson of Queens University Belfast, describes the factors to be considered at each of these steps and compaers workflows from Qiagen and Bioo Scientific/PerkinElmer for small RNA sequencing from serum and plasma samples.
The workflow from Bioo Scientific involves magnetic bead-based RNA purification followed by library preparation using adapters with randomized ends to reduce ligation bias, while the Qiagen workflow involves column-based purification of RNA followed by library preparation utilizing unique molecular identifiers for PCR bias correction. Dr. Simpson discusses metrics such as mapping rate, adapter-dimer contamination, and diversity of miRNAs detected.
Dr. Simpson also discusses the effectiveness of the alternative chemistries and randomized adapters employed by Bioo Scientific to reduce ligation bias. For example, the correction of PCR bias by use of unique molecular identifiers in the Qiagen workflow had little effect upon relative miRNA quantification, suggesting that reduction of ligation bias is a more effective strategy to reduce bias in small RNA library preparation.