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.
The novel ‘K’ DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing that maintains representation of microbial populations in a sample, with the added benefits that the K method reduces hands-on time by up to 20 x for 96 sample preparations.READ MORE