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

Sabrina Shore,Michelle P Salcedo,Gerald Zon,Alexandre Lebedev,Jordana M Henderson,Richard I Hogrefe,Anton P Mccaffrey,Natasha Paul,Ramamurthy Mahalingam

doi:10.1371/journal.pone.0167009

Sabrina Shore, Michelle P Salcedo + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0167009

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Abstract

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.

Highlights

next-generation sequencing (NGS) is a powerful tool that yields vast amounts of sequence data and allows the analysis of many samples in parallel
We designed matrices that combined one of the modified 3’ adapters with each of the modified 5’ adapters and vice versa producing 256 combinations of modifications (S1 Table). These combinations of modifications were interrogated with the following criteria in mind: 1) ability to ligate to an unmodified RNA library; 2) inhibition of 5 ́ and 3 ́ adapter ligation to prevent adapter dimer; and 3) ability of reverse transcriptase enzymes to read through modifications, when separated by an RNA insert, to form an unmodified cDNA for downstream PCR
With 1 nanogram total RNA input using CleanTag adapters, we found less than 1% of reads lost to adapter dimer while sustaining similar levels of mappable reads compared to the higher inputs (S2 Table)

Summary

Introduction

NGS is a powerful tool that yields vast amounts of sequence data and allows the analysis of many samples in parallel. Correlation with genetic databases allows identification of novel biomarkers for better diagnosis and more personalized treatment. Next-generation sequencing can be used to examine DNA or RNA for whole genome or transcriptome analysis, respectively. Various techniques such as Chromatin Immunoprecipitation (ChIP), Cross-linking Immunoprecipitation (ClIP), and RNA Immunoprecipitation Sequencing (RIP-Seq) isolate protein/nucleic acid complexes and sequencing of the partitioned nucleic acids allows cataloging of molecular interactions. Transcriptome analysis can be further divided into long coding RNA (messenger RNA or mRNA), long non-coding RNA (lncRNA), or small RNA (sRNA). SRNA are < 500 nucleotides (nt) and consist of a variety of sRNA. Transcriptome analysis can be further divided into long coding RNA (messenger RNA or mRNA), long non-coding RNA (lncRNA), or small RNA (sRNA). sRNA are < 500 nucleotides (nt) and consist of a variety of sRNA

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