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Published on: September 16, 2019
Method for improved Illumina sequencing library preparation using NuGEN Ovation RNA-Seq System.
Steven R Head1, H Kiyomi Komori, G Traver Hart
1Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, CA 92040, USA. shead@scripps.edu
Biotechniques
|April 14, 2011
Summary
Treating amplified cDNA with single-strand endonuclease S1 significantly improves next-generation sequencing library yields. This method effectively cleaves hairpin structures, enhancing downstream library construction efficiency for RNA-Seq.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Next-generation sequencing (NGS) library preparation using the NuGEN Ovation RNA-Seq System can produce cDNA with hairpin structures.
- These ssDNA hairpin structures impede crucial downstream steps like end repair, A-tailing, and adapter ligation.
- Inefficient library construction reduces overall sequencing yield and data quality.
Purpose of the Study:
- To investigate methods for improving the efficiency of NGS library preparation.
- To address the challenge of hairpin structures in NuGEN-amplified cDNA.
- To enhance sequencing library yields for RNA-Seq applications.
Main Methods:
- Utilized the NuGEN Ovation RNA-Seq System for cDNA library preparation.
- Applied single-strand endonuclease S1 treatment to NuGEN-amplified cDNA.
- Performed downstream library construction steps for Illumina GAIIx sequencing.
- Quantified sequencing library yields before and after S1 treatment.
Main Results:
- Treatment with single-strand endonuclease S1 increased sequencing library yields by 4- to 6-fold or greater.
- S1 treatment effectively cleaved hairpin structures resistant to standard end-repair enzymes.
- Improved efficiency in end repair, A-tailing, and adapter ligation was observed.
Conclusions:
- Single-strand endonuclease S1 treatment is a highly effective strategy to enhance NGS library preparation.
- Cleavage of hairpin structures by S1 significantly boosts sequencing library yields.
- This method offers a valuable improvement for RNA-Seq library construction workflows.
