EliGene® kits for Next Generation Sequencing

EliGene® NGS kits are used for one or more multiplex PCR reactions resulting in amplification of target regions of one sample (Figure 2A). The resulting amplicons are barcoded with MID sequences, and p7, p5 adaptors required for Illumina MiSeq Sequencing (Figure 2B). Each amplicon library is subsequently purified from small residual DNA fragments shorter than 150bp and the DNA concentration is determined using a Real-Time PCR. Next, these purified and individually tagged amplicon libraries are pooled equimolar, resulting in an amplicon pool or sequencing sample, which is sequenced using a NGS instrument Illumina for massively parallel sequencing (MPS) according to the manufacturer's instructions. The resulting sequence reads are subsequently analysed by comparison with the reference sequence of the targeted gene(s) to identify variant positions. For amplification is used a ready-to-use mastermix containing a hot-start DNA polymerase The positions of the individual custom sequencing primers supplied by EliGene® NGS kit as well as the Illumina sequencing reagents are indicated and clarified in Figure 2C.

Figure 2. Layout of the process resulting in multiplex PCR of target regions (A), incorporation of MIDs and p7 and p5 adaptors (B) and structure of resulting product after the sequencing on Illumina MiSeq instrument (C)