NEXTFLEX® Small RNA-seq v3 Kits

  • 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
  • Automated on the Sciclone® G3 NGS/NGSxSciclone® G3 NGSx iQ™,  and the Zephyr® G3 NGS workstation
For research use only. Not for use in diagnostic procedures.

  • NOVA-5132-05


  • NOVA-5132-06

    48 BARCODES | 48 RXNS

  • NOVA-5132-22/23/24/25

    UDI Barcodes 48 rxns each

  • NOVA-5132-26/27/28

    UDI Barcodes 48 rxns each

  • NOVA-5132-29

    UDI Barcodes 48 rxns each

  • NOVA-5132-18/19

    UDI Barcodes 96 rxns each

  • NOVA-5132-20/21

    UDI Barcodes 96 rxns each

  • NOVA-513121

    8 RXNS

  • NOVA-513122

    48 RXNS

  • NOVA-513123

    96 RXNS

Gel-Free Small RNA Library Prep with Randomized Adapters for Reduced Bias

Learn about the new NEXTFLEX® Small RNA-Seq Kit v4 delivering a simplified miRNA low input workflow with improved discovery, even with low input samples.

The NEXTFLEX® Small RNA-Seq Kit v3 uses patented and patent-pending technology to provide a reduced-bias small RNA library preparation solution for Illumina® sequencing platforms with gel-free or low-input options. Our approach to reducing ligation-associated bias involves the use of adapters with randomized bases at the ligation junctions, resulting in greatly decreased bias in comparison to standard protocols. This reduction in bias results in data that more accurately represents abundances of small RNAs in the starting material. In addition, reduction of bias allows more miRNAs to be detected with fewer total reads, increasing efficiency and reducing cost for small RNA sequencing.

PAGE purification, required for traditional small RNA library prep, is tedious, time consuming, limits throughput, and prevents start-to-finish automation. The NEXTFLEX® Small RNA-Seq Kit v3 allows for gel-free small RNA library preparation when starting with ≥200 ng of total RNA. Libraries prepared with the NEXTFLEX® Small RNA-Seq kit v3 have a high proportion of reads mapping to miRNAs (Fig. 1). For low input samples, the new NEXTFLEX® Small RNA-seq kit v4 enables gel-free library prep from input as low as 1 ng of total RNA with improved discovery rates.

The NEXTFLEX® small RNA-seq v3 kit with UDIs uses the same popular chemistry of the NEXTFLEX small RNA-seq kit v3 and improved 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,

Sequencing depth vs miRNAs detected
The NEXTFLEX® Small RNA-Seq kit V3 “consistently performed well with respect to enrichment of miRNA mapping reads in biofluids and tissues” and “exhibited a relatively low quantification bias”.
Coenen-Stass, A.M.L., et al. (2018) Evaluation of methodologies for microRNA biomarker detection by next generation sequencing. RNA Biology. , 15: 8. 15:8, 1133-1145.

Low Input Small RNA Library Prep for Illumina® Sequencing

The adapter-dimer reduction technology incorporated into this kit also allows low input library preparation. Library preparation with as little as 1 ng of total RNA is possible as additional PCR cycles can be performed without adapter-dimer products dominating the final library. Fig. 2 illustrated that expression values are reproducible across different sample inputs.

Low input small RNA library prep with as little as 1 ng of total RNA

Independent Analysis of Small RNA Library Prep Solutions

Seven independent studies were published in 2018 and 2019 comparing small RNA sequencing data obtained using commercially available small RNA-seq library prep kits. The NEXTFLEX® Small RNA-Seq kit was recommended in every study because of the low bias and consistent results obtained using the kit.

Chu, C. P., & Nabity, M. B. (2019). Comparison of RNA isolation and library preparation methods for small RNA sequencing of canine biofluids. Veterinary Clinical Pathology. doi:10.1111/vcp.12743

Coenen-Stass, A.M.L., et al. (2018) Evaluation of methodologies for microRNA biomarker detection by next generation sequencing. RNA Biology. 15: 8. 15:8, 1133-1145. doi: 10.1080/15476286.2018.1514236.

Dard-Dascot, C., et al. (2018) Systematic comparison of small RNA library preparation protocols for next-generation sequencing. BMC Genomics 19(118), doi:10.1186/s12864-018-4491-6.

Giraldez, M. D., Spengler, R. M., Etheridge, A., Godoy, P. M., Barczak, A. J., Srinivasan, S., . . . Tewari, M. (2018). Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling. Nature Biotechnology. doi:10.1038/nbt.4183.

Ku, A., Ravi, N., Yang, M., Evander, M., Laurell, T., Lilja, H., & Ceder, Y. (2019). A urinary extracellular vesicle microRNA biomarker discovery pipeline; from automated extracellular vesicle enrichment by acoustic trapping to microRNA sequencing. Plos One, 14(10). doi: 10.1371/journal.pone.0224604.

Wright, C., Rajpurohit, A., Burke, E. E., Williams, C., Collado-Torres, L., Kimos, M., . . . Shin, J. H. (2018). Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods. bioRxiv 445437. doi:10.1101/445437.

Yeri, A., et al. (2018) Evaluation of commercially available small RNASeq library preparation kits using low input RNA. BMC Genomics 201819:331. doi: 10.1186/s12864-018-4726-6.

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.

Small RNA-Seq Automation Compatibility

The NEXTFLEX® small RNA-seq v3 automation kit with UDIs and the NEXTFLEX® Small RNA-Seq Kit V3 is designed for easy migration onto automated liquid handling platforms. Currently methods are available for the Sciclone® NGS/NGSx and the Zephyr® G3 NGS workstation. Components of the NEXTFLEX® small RNA-seq v3 automation kit with UDIs have been optimized and validated on these liquid handling platforms, and the UDI barcoded primers are conveniently arrayed in plated format for easy automation.

Download the Sciclone® NGS and NGSx Workstation Automation Guide for the NEXTFLEX Small RNA-Seq Kit V3.

Download the Zephyr® G3 NGS Workstation Automation Guide for the NEXTFLEX Small RNA-Seq Kit v3.

For more information contact [email protected].

Enabling 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. The barcoded primers are plated in column format.

Eight barcoded primers are included in the eight reaction NEXTFLEX® Small RNA-Seq Kit v3 and forty-eight barcoded primers are included in the forty-eight reaction NEXTFLEX® Small RNA-Seq Kit v3. These barcoded primers included in these kits are not UDI.

For research use only. Not for use in diagnostic procedures.