Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

<div><p>For most sample types, the automation of RNA and DNA sample preparation workflows enables high throughput next-generation sequencing (NGS) library preparation. Greater adoption of small RNA (sRNA) sequencing has been hindered by high sample input requirements and inherent ligation side products formed during library preparation. These side products, known as adapter dimer, are very similar in size to the tagged library. Most sRNA library preparation strategies thus employ a gel purification step to isolate tagged library from adapter dimer contaminants. At very low sample inputs, adapter dimer side products dominate the reaction and limit the sensitivity of this technique. Here we address the need for improved specificity of sRNA library preparation workflows with a novel library preparation approach that uses modified adapters to suppress adapter dimer formation. This workflow allows for lower sample inputs and elimination of the gel purification step, which in turn allows for an automatable sRNA library preparation protocol.</p></div

Investigation of modified adapter combinations at lower RNA inputs.

<p>Brain total RNA at 1000 or 100 ng input was tested with candidate modified adapters in a full library preparation workflow. The modified 3´ adapter was MP (n-1) and the modified 5´ adapter was either 2´ OMe (n) or 2´ OMe (n-2). Agarose gel analysis of the product from 12 cycles of PCR. No adapter dilutions were made.</p

A comparison of small RNA library preparation workflows.

<p>A) The traditional approach with unmodified adapters which results in tagged library and adapter dimer. B) The modified adapter approach which results in primarily tagged library.</p

Comparison of NGS data between gel purified and bead purified samples within a CleanTag workflow.

<p>Libraries prepared with CleanTag small RNA library prep kit and human brain total RNA input. PCR samples were purified by gel extraction or 2-step AMPure XP bead-based protocol. Data analysis was performed with Geneious.</p

Agarose gel analysis of PCR purified libraries with various total RNA inputs at 10ng.

<p>Libraries prepared with CleanTag workflow. UHR is Universal Human Reference RNA.</p

Next-generation sequencing run comparing top modified adapter combinations.

<p>Libraries prepared with unmodified or modified (MP(n-1)) 3´ adapter and unmodified or various modified 5´ adapter (1 = OMe(n), 2 = MP (n-1), or 3 = Ps(n)) using a pool of 963 synthetic miRNA (Miltenyi) following CleanTag library preparation protocol. Data analysis performed by TSRI. A) Agarose gel analysis of crude library PCR products. Sequencing Data: B) Average number of adapter dimer reads, C) Average number of filtered reads.</p

Comparison of crude and bead purified libraries using CleanTag or TruSeq small RNA library prep kit.

<p>Bioanalyzer traces of libraries prepared using 1000 ng human total brain RNA input. Crude or AMPure XP purified PCR products with A) TruSeq small RNA library preparation kit, or B) CleanTag small RNA library preparation kit.</p

Optimization of the 3´ adapter ligation step.

<p>Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.</p

Screen for the best combination of modified adapter pairs for suppression of adapter dimer.

<p>Top combinations of modified adapters were tested in a full CleanTag library prep workflow from ligation to RT-PCR for dimer suppression. 0.7 ng Let-7d-3p (NNN) synthetic miRNA input. Samples run on a 4% agarose gel stained with ethidium bromide. Best combinations are shown in red boxes. U = unmodified.</p

NGS data comparison between CleanTag and TruSeq Small RNA Library Preparation Kit.

<p>Libraries prepared with TruSeq Small RNA Library Preparation Kit or CleanTag workflow with total human brain RNA input and gel purification. Samples sequenced on a HiSeq 2500 SR, 1x 100bp. Human total brain RNA at A) 100 ng, or B) 10 ng input. Data analysis performed using Geneious. Statistical analysis performed with GraphPad-One way ANOVA Turkeys multiple comparison test.</p