The SeqPlex-I WTA kit allows amplification of small quantities of reverse transcribed RNA or degraded RNA for direct input onto Illumina® next-generation sequencing (NGS) flow cells. The SeqPlex-i process is comprised of three steps: Pre-amplification/Library Synthesis, Amplification 1 and Amplification 2
Step 1: In the Pre-amplification/Library Synthesis step using the (Library Preparation Reagents), the template RNA is reverse transcribed using primers composed of a semi-degenerate 3′- and universal 5′-ends. As polymerization proceeds, displaced and RNaseH generated single strands serve as new templates for additional primer annealing and extension producing random, overlapping cDNAs flanked by a universal primer (5′) and primer complement (3′) sequence.
Step 2: In the Amplified Library Synthesis step (using the Amplification 1 Reagents), products from pre-amplification/library synthesis are amplified by single primer PCR via the universal end sequence. These amplification products typically range from 200 to 500+ base pairs.
Note: Amplicons from degraded RNA, such as Formalin Fixed Paraffin Embedded (FFPE), are typically shorter and dependent upon the length of the starting RNA.
Step 3: In the Sequencing Library Synthesis step (using Amplification 2 Reagents), single primer amplicons from amplification 1 are converted to dual Illumina® primer PCR products ready for purification, quantification, and Illumina® NGS.
SeqR-i RNA Amplification Kit has been used for whole transcriptome amplification.
Features and Benefits
- Amplifies fragmented/extremely small quantities of total RNA: Fragmented or intact RNA from all sources including FFPE and RIP are easily amplified by random priming technology.
- Semi-degenerate library primer design ensures more complete transcriptome coverage and efficient priming
- Fewer Steps: No need to fragment cDNA before sequencing
- High-efficiency: Amplifies ds-cDNA in 8 hours or less
- Cost-effective: No longer requires an additional NGS library prep step
- Compatible with Illumina® next generation sequencing
1) RNA Handling Technique
a) The reagents in this kit have been tested to assure that RNases are absent.
b) The user, however, must protect the integrity of experimental results by wearing basic protective equipment, including gloved hands and lab coat.
c) All reagent transfers throughout this procedure should be performed in a laminar flow hood or dedicated clean room.
d) Frozen RNA samples should be thawed on ice.
2) A 20 μL Amplification 2 reaction will produce >100 ng of amplified double-stranded cDNA when starting with 100 pg to 5 ng of high-quality RNA. Higher input quantities and higher quality RNA template generally result in increased yields. For damaged RNA, such as from FFPE, 1–50 ng input RNA is recommended.
3) The dual index adapter primers (AP100) provided in this kit will only work for one sample. If pooling samples for sequencing is required, the user must provide additional index primer sets. See example index primer sequences on page 2 of the technical bulletin.
Illumina is a registered trademark of Illumina, Inc.
The SeqPlex-I DNA Amplification Kit for whole genome amplification (WGA) is for R&D use only. Not for drug, household, or other uses.