Precision small RNA library kit for Illumina® NGS systems using automated library preparation

QIAseq miRNA enables Sample to Insight®, precision next-generation sequencing (NGS) of mature miRNAs on Illumina NGS instruments. This highly optimized solution facilitates both enhanced differential expression analysis using integrated unique molecular indices (UMIs) and novel discovery of miRNA from cells, tissues, and biofluids. The required amount of template for a single QIAseq miRNA sequencing reaction can range from 500 ng to as little as 1 ng of purified total RNA.

In recent years, NGS has emerged as a highly advanced research tool for both high-throughput miRNA expression analysis and novel miRNA discovery. Among commercially available solutions, QIAseq miRNA defines a new generation of small RNA sequencing products and includes several distinct features not found in other sequencing kits. The standard QIAseq miRNA procedure does not require gel purification, excision, and elution, which substantially reduces the required hands-on time and noticeably shortens the length of the whole workflow. Proprietary methodology utilizing modified oligonucleotides efficiently prevents adapter– dimerization in the sequencing library and the highly optimized reaction chemistry virtually eliminates biases and background contaminants, facilitating the preparation of robust, miRNAspecific libraries. The kit also integrates UMIs into the reverse transcription process, enabling unbiased and accurate miRNome-wide quantification of mature miRNAs by NGS. Should a library fail presequencing quality control (QC), in-line controls are included in the library generation procedure to allow the use of real-time PCR for fast and efficient troubleshooting. Both primary and secondary data analysis solutions have been developed to facilitate rapid and robust UMI counting, miRNA mapping, and differential expression analysis. Overall, QIAseq miRNA offers an unrivaled Sample to Insight solution for differential expression analysis and discovery of novel miRNAs using NGS.

Principle and procedure
Mature miRNAs are naturally occurring, 22-nucleotide, noncoding RNAs that mediate post-transcriptional gene regulation. Unlike most cellular RNAs, mature miRNAs possess both a 3' hydroxyl group and a 5' phosphate group. This allows adapters to be specifically ligated to both the 3' end and 5' end of miRNAs enabling universal reverse transcription and library preparation of mature miRNAs, while minimizing the background from other RNA species.
The QIAseq miRNA Library Auto Kit (384) with the QIAseq miRNA 96 Index IL Auto A (384) provides enough reagents for automated library preparation of up to 384 samples and multiplexing of up to 96 samples per flow cell lane on Illumina NGS instruments. Universal cDNA synthesis and library preparation of miRNA In an unbiased reaction, adapters are ligated sequentially to the 3' and 5' ends of miRNAs. Subsequently, universal cDNA synthesis with UMI assignment, cDNA cleanup, library amplification, and library cleanup are performed. Proprietary methodology using modified oligonucleotides virtually eliminates the presence of adapter–dimers in the sequencing library, effectively removing a major contaminant often observed during sequencing. Additionally, the kit is designed to also minimize the presence of hY4 Y RNA, which is often observed in high levels in serum and plasma samples.

NGS on Illumina NGS systems

miRNA sequencing libraries prepared with the QIAseq miRNA Library Automation Kit can be sequenced using an Illumina NGS system (MiSeq® Personal Sequencer, NextSeq 500/550, HiSeq® 1000, HiSeq 1500, HiSeq 2000, HiSeq 2500, HiSeq 4000, NovaSeq™ 6000, and GAIIx). QIAseq miRNA Library Auto Kit (cat. no. 331509) derived libraries require 75 bp single reads. It is recommended to allocate 5–10 million reads per sample. A 50 bp single read protocol can be used if there is no desire to include the UMIs. If a 50 bp single read protocol is used, primary data analysis cannot be performed using the QIAseq miRNA Primary Data Analysis pipeline. To use the QIAseq miRNA Primary Data Analysis pipeline, UMIs must be sequenced.

Integrated reaction controls
The QIAseq miRNA Library Kit contains integrated reaction controls to monitor the 3' ligation, 5' ligation, and reverse transcription (Table 3). Together, the controls monitor critical steps of the workflow. If library QC (Protocol: miRNA Library Presequencing QC) is unsuccessful (if, for instance, no peak is observed during Bioanalyzer® analysis), these controls can be assessed using real-time PCR. This helps to determine if the absence of a library is due to a technical or sample issue (see Appendix B: Real-time PCR Troubleshooting) and at which step the library preparation failed.