Improved Small RNA Library Preparation Workflow for Next-Generation Sequencing
Poster Mar 22, 2015
Sabrina Shore, Jordana Henderson, Anton McCaffrey, Gerald Zon, Richard Hogrefe
Next generation sequencing (NGS) is used to analyze microRNAs (miRNAs), small non-coding RNAs that are important therapeutic targets and diagnostic markers. Commercially available small RNA sequencing library preparation kits require large inputs (>100 ng) and a laborious gel purification step, which is not amenable to automation. Additionally commercial kits are hindered by adapter dimer formation, where 5΄ and 3΄ adapters ligate without an intervening RNA insert. Adapter dimers preferentially amplify during PCR amplification. This is exacerbated at low RNA inputs where adapter dimers can greatly diminish usable sequencing reads. Current methods for adapter dimer suppression are insufficient to allow sequencing of very low input amounts and require a gel purification step to remove adapter dimers. In contrast, we describe an optimized workflow which suppresses adapter dimers, allows for RNA inputs down to 1 ng and eliminates the need for a gel purification step. Our workflow introduces chemically modified adapters that allow efficient library ligation while suppressing adapter dimer formation. TriLink’s modified adapter workflow allows RNA inputs as low as 1 ng with less than 1 % adapter dimer reads when gel purified. Furthermore, the improved workflow using magnetic bead-based size selection allows for automation which was not previously possible.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE