Small RNA Library Prep with Randomized Adapters for Reduced Sequencing Bias and Unique Dual Indexes to Mitigate Index Hopping

The NEXTFLEX® small RNA-seq v3 kit with UDIs uses the same popular chemistry of the NEXTFLEX small RNA-seq kit v3, which incorporates technologies that reduce small RNA ligation bias, increase ligation efficiency, reduce adapter-dimer formation, and build in a new dimension of data quality, integrity, and accuracy with the integration of Unique Dual Indexes (UDIs) for Illumina® sequencing on the MiSeq®, HiSeq® 2000/2500, and NovaSeq® platforms. The addition of UDI barcodes for small RNA sequencing allows confident multiplexing of up to 192 samples, all while mitigating the risk of index hopping and spread of signal that can occur on a patterned flow cell. The purity of each index sequence is validated by sequencing for high confidence in the resulting sequencing data quality.

The NEXTFLEX® small RNA-seq v3 kit with UDIs is compatible with cell-free RNA, such as RNA isolated from plasma. Users who wish to deplete the abundant tRNA fragments and YRNA fragments found in many types of cell-free RNA should use the NEXTFLEX® tRNA/YRNA blocker (not included in kit).

Completely Automated Small RNA-seq Library Prep

The NEXTFLEX® small RNA-seq v3 automation kit with UDIs has been designed for automation on the PerkinElmer® Sciclone® G3 NGS/NGSx, Sciclone® G3 NGSx iQ™, and Zephyr® G3 NGS workstations. Components of the kit have been optimized and validated on these liquid handling platforms, and the UDI barcoded primers are conveniently arrayed in plated format for easy automation.

Enabling Unprecedented High-Level Multiplexing for Small RNA Sequencing Efficiency

96 barcoded primers (barcodes 1 – 96 or barcodes 97-192) are included in the 96 reaction NEXTFLEX® small RNA-seq v3 kits with UDIs. For Illumina® sequencing on the MiSeq®, HiSeq® 2000/2500, and NovaSeq® platforms, the barcoded primers behave as unique dual indexes (UDIs), while when used on the Illumina® MiniSeq®, HiSeq® 3000/4000, HiSeqX®, and NextSeq® platforms, the barcoded primers behave as single index barcodes.

Barcoded primers are plated in column format.

More miRNA Discovery by Blocking tRNA/YRNA Fragments

Including the NEXTFLEX® tRNA/YRNA blockers into your small RNA-sequencing workflow can help minimize YRNA and tRNA fragments from making their way into your library, which consequently increases miRNA reads. tRNA and YRNA fragments, though they may have unexplored roles as potential biomarkers, are abundant in sample types like biofluids. For researchers, however, who are primarily interested in other small RNA species like miRNAs, reads lost to these abundant tRNA and YRNA fragments in their sample will inevitably lead to a lower number of reads mapping to their RNA species of interest. Coupling the NEXTFLEX® tRNA/YRNA blockers with the NEXTFLEX® small RNA sequencing chemistry and data security of UDIs allows users to maximize the patented and patent-pending technology for ligase-associated bias reduction, which enables superior miRNA discovery for the same sequencing depth.

Simplify Small RNA-Seq Bioinformatic Analysis

The NEXTFLEX® sRNA Tool, which is provided at no-cost, simplifies the bioinformatic analysis of small RNA sequencing datasets delivering information on read profiles, insert sizes for each sample, and normalized heatmaps. Simply upload sequence data files (fastq.gz format) and choose a reference organism from the drop-down menu. Once the analysis is completed, a notification email is delivered with a link to the results, which can be visualized within the tool or downloaded for further analysis.

Features

• Greater discovery/detection rate reduces sequencing costs

• Randomized adapter technology reduces ligation bias and increases accuracy

• As little as 200 ng input starting material for complete automation

• Barcoded primer sets allows multiplexing of up to 384 samples

• Unique dual index barcoded primer set mitigates index hopping on the Illumina® NovaSeq®, MiSeq®, and HiSeq® 2000/2500 platforms

• Individually sequence-validated indices ensure confidence in purity

• Automation kits available for the PerkinElmer® Sciclone® G3 NGS/NGSx, Sciclone® G3 NGSx iQ™,  and the Zephyr® G3 NGS workstation saves time and resources

Kit Specs

KIT CONTENTS

• NEXTFLEX® 3’ 4N Adenylated Adapter

• NEXTFLEX® 3’ Ligation Buffer

• NEXTFLEX® 3’ Ligation Enzyme Mix

• NEXTFLEX® Adapter Depletion Solution

• NEXTFLEX® Adapter Inactivation Buffer

• NEXTFLEX® Adapter Inactivation Enzyme

• NEXTFLEX® 5’ 4N Adapter

• NEXTFLEX® 5’ Ligation Buffer

• NEXTFLEX® 5’ Ligation Enzyme Mix

• M-MuLV Reverse Transcriptase

• NEXTFLEX® RT Buffer

• NEXTFLEX® UDI Barcoded Primer 1 – 96 or 97 – 192*

• NEXTFLEX® Small RNA PCR Master Mix

• microRNA Control

• Resuspension Buffer

• Nuclease-free Water

• NEXTFLEX® Adapter Depletion Solution

• NEXTFLEX® Cleanup Beads

* Barcoded primers are plated in column format.