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.
The SeqPlex-I DNA Amplification Kit for whole genome amplification (WGA) is for R&D use only. Not for drug, household, or other uses.