Improved Small RNA Library Preparation Workflow for Next Generation Sequencing

Abstract
Next generation sequencing (NGS) can be 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 relative to the library during PCR amplification. This is exacerbated at low RNA inputs where adapter dimers can greatly diminish usable sequencing reads. We describe an optimized small RNA library preparation workflow which suppresses adapter dimers, allows for RNA inputs as low as 1 ng and eliminates the need for gel purification. Chemically modified adapters and an optimized protocol were employed to suppress adapter dimers while still allowing for efficient library ligation. Library preparation with modified adapters was compared to the Illumina TruSeq® Small RNA Sample Prep Kit. Non-gel purified samples were purified with the Agencourt® AMPure® XP Kit. Samples were sequenced on an Illumina HiSeq™. Our modified adapter workflow was benchmarked against the TruSeq® Kit at 100 and 10 ng inputs. The modified adapter workflow allows RNA inputs as low as 1 ng and generates less than 1% adapter dimer reads when gel purified (Table 1). In contrast, the TruSeq® Kit yields 14% and 51% adapter dimer reads at 100 and 10 ng inputs, respectively. TriLink’s modified adapter workflow with magnetic bead-based size selection yields less than 10% dimer for all input levels, while the TruSeq® Kit results in a minimum of 14% dimer reads at the highest input level. TriLink’s modified adapter workflow improves small RNA library preparation by significantly reducing adapter dimer formation. In fact, our improvements allow for sequencing from 1 ng of total RNA without compromising valuable sequencing reads, which was previously not feasible. TriLink’s workflow with magnetic bead-based size selection, an automatable technique, results in lower amounts of dimer reads than current methods using gel purification. Replacement of gel purification with an automatable purification step results in less hands-on time, better reproducibility and higher throughput. The modified adapter workflow surpasses other currently available technologies and provides significant improvement to small RNA NGS.