The SeqPlex™-I WGA kit allows amplification of small quantities of DNA or from degraded/highly fragmented DNA 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 DNA is replicated using primers comprised of semi-degenerate 3′- ends with universal 5′- ends. As polymerization proceeds, displaced single strands serve as new templates for additional primer annealing and extension producing random, overlapping replicons 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.
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.
SeqPlex™-I WGA Kit has been used for whole genome amplification.
Features and Benefits
- Amplifies fragmented/extremely small quantities of DNA: Yields from ChIP or FFPE, which are 200-500 bp are easily amplified by Random priming technology.
- Facilitates sequencing from as little as 100 pg of DNA
- Enhanced primers for complete genome coverage, minimal sequence bias, and amplicon size ideal for next generation sequencing (NGS)
- Cost-effective: No longer requires an additional NGS library prep step
- Compatible with Illumina® next-generation sequencing
- 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 DNA. Higher input quantities and higher quality DNA template generally result in increased yields. For damaged DNA, such as from FFPE, 1–50 ng input DNA is recommended.
- The dual index adapter primers (AP100) provided in this kit will only work for one sample. If pooling of 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.
SeqPlex is a trademark of Sigma-Aldrich Co. LLC
The SeqPlex-I DNA Amplification Kit for whole genome amplification (WGA) is for R&D use only. Not for drug, household, or other uses.