Gel-free, Combined Small RNA and mRNA Library Prep with Randomized Adapters to Reduce Ligation Bias and UDIs to Prevent Sample Mis-assignment on Illumina® Platforms

The NEXTFLEX® Combo-Seq™ Kit enables the user to generate combined mRNA and small RNA libraries in a single workflow using 5 ng – 100 ng of total RNA inputs, without requiring upfront costly and tedious rRNA depletion or poly(A) selection. The kit utilizes patented and patent-pending technology that allows processing of mRNA fragments and small RNA for library construction. Like the best-in-class, NEXTFLEX® small RNA-seq kit v3, the NEXTFLEX® Combo-Seq™ mRNA/miRNA library preparation kit utilizes adapters with randomized ends to greatly reduce bias compared to standard protocols. This allows a more accurate representation of small RNA in the starting material and cost-saving due to efficient sequencing. Adapter-dimer reducing techniques built into the protocol enables gel-free workflow from inputs as low as 5 ng of total RNA. As a result, the kit is streamlined and Automation-friendly from start-to-finish. Libraries with robust yield can be prepared within one day using a manual or automated protocol.

Automation Compatibility

The kit is designed for seamless automation on the PerkinElmer® Sciclone® G3 NGS/NGSx workstations. 

Facilitating Both High- and Low-throughput Needs for Illumina® Multiplexing

Eight UDI barcoded primers are included in the small reaction size NEXTFLEX® Combo-Seq™ kit and forty-eight UDI barcoded primers are included in the large reaction size kit. UDIs read as single index reads on certain instruments, including the Illumina® MiniSeq®, NextSeq®, HiSeq® 3000 or HiSeq® 4000 platforms.

Features

• Combined library prep workflow for both small RNA and mRNA sequencing

• Completely gel-free protocol from inputs as low as 5 ng

• Compatible with total RNA inputs, no poly(A) selection or rRNA depletion required

• Complete kit solution, including cleanup/size selection beads and barcodes*

• Automated on the PerkinElmer Sciclone® G3 NGS/NGSx workstations

Kit Specs

Figure 1. NEXTFLEX® Combo-Seq mRNA/miRNA kit discovers ~60% more transcripts compared to Competitor N’s mRNA-seq workflow. Libraries were generated from 20 ng of Biochain Human Universal RNA (R4234565) using the NEXTFLEX® Combo-Seq mRNA/miRNA Kit and Competitor N’s directional mRNA-seq workflow. Sequencing libraries were downsampled to 10M reads and processed using the exceRpt pipeline (https://hub.docker.com/r/rkitchen/excerpt). Pipeline output files were used to enumerate ”transcripts discovered.” Transcripts were only marked “discovered” if >= 3 reads mapped to a given transcript and if each read aligned in the appropriate direction (sense for the NEXTFLEX® kit and antisense for Competitor N’s kit). For “long RNA,” transcripts were counted using the “geneLevel” read counts files. In the figure, pink indicates transcripts which were detected by both workflows, whereas purple and red refer to NEXTFLEX® and Competitor N, respectively.

Figure 2. The NEXTFLEX® Combo-Seq™ mRNA/miRNA kit gives higher confidence in strand calls compared to Competitor N’s directional mRNA-seq workflow. Reads were mapped to synthetic ERCC’s to determine orientation. NEXTFLEX® chemistry has >99.9% directionality whereas Competitor N is only ~99% directional (data not shown), which allows for higher directionality/ strandedness and lower rate of false-positives regarding directionality.

Figure 3. The NEXTFLEX® Combo-Seq™ mRNA/miRNA kit gives more accurate representation of miRNAs than Competitor N’s small RNA-seq kit. In order to assess bias, libraries were generated from a pool of 963 equimolarly pooled miRNAs using the NEXTFLEX® Combo- Seq™ mRNA/miRNA kit and a commercially available small RNA-seq kit from Competitor N. Individual miRNAs were binned into categories based on their representation in the sequencing library in terms of fold-change from expected. With the NEXTFLEX® Combo-Seq™ mRNA/miRNA kit, only 5.8% of miRNAs deviate more than 10-fold from the expected value, compared with 30.1% of miRNAs with Competitor N.

KIT CONTENTS

• NEXTFLEX® Anchored Oligo(dT) Primer

• NEXTFLEX® RT/RNase H Annealing Buffer

• NEXTFLEX® RT/RNase H Reaction Buffer

• NEXTFLEX® Combo-Seq™ Reverse Transcriptase

• NEXTFLEX® Thermostable RNase H

• NEXTFLEX® PAP Buffer

• NEXTFLEX® Poly(A) Polymerase

• NEXTFLEX® tRNA/YRNA Blocker

• Adapter Depletion Solution

• NEXTFLEX® Combo-Seq™ 5’ Ligation Buffer

• NEXTFLEX® Combo-Seq™ 5’ 4N Adapter

• NEXTFLEX® 5’ Ligation Enzyme Mix

• NEXTFLEX® PAP Buffer

• NEXTFLEX® Poly(A) Polymerase

• NEXTFLEX® Combo-Seq™ RT Buffer

• NEXTFLEX® PAP Buffer

• NEXTFLEX® UDI Barcoded Primer Mix

• NEXTFLEX® PCR Master Mix

• Nuclease-free Water

• Resuspension Buffer

• NEXTFLEX® Cleanup Berads

REQUIRED MATERIALS NOT PROVIDED

• 5 ng – 100 ng total RNA in up to 11 μL Nuclease-free Water

• Human Universal RNA (Biochain # R4234565) or other total RNA, as a control (optional)

• Isopropanol

• 80% Ethanol, freshly prepared

• 10, 20, and 200 μL pipettes

• 10, 50, and 200 μL multichannel pipettes

• RNase-free barrier pipette tips

• 96 well PCR Plate Non-skirted (Phenix Research # MPS-499) or similar

• Nuclease-free PCR strip tubes with caps (Phenix Research # MPC-425Q)

• Nuclease-free 1.7 mL microcentrifuge tubes

• Thermal cycler

• Vortex

• Microcentrifuge

• Magnetic Stand -96 (Ambion # AM10027 or similar)