H24
The Illumina NGS workflow includes 4 basic steps :1) Sample Preparation For library construction, DNA/RNA is extracted from a sample. After performing quality control(QC), qualified samples proceed to library construction. 2) Library ConstructionThe sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followedby 5' and 3' adapter ligation. Alternatively, "tagmentation" combines the fragmentation and ligationreactions into a single step that greatly increases the efficiency of the library preparation process. Adapter ligated fragments are then PCR amplified and gel purified. 3) Sequencing For cluster generation, the library is loaded into a flow cell where fragments are captured on a lawnof surface-bound oligos complementary to the library adapters. Each fragment is then amplifiedinto distinct, clonal clusters through bridge amplification. When cluster generation is complete, thetemplates are ready for sequencing. Illumina SBS technology utilizes a proprietary reversible terminator-based method that detectssingle bases as they are incorporated into DNA template strands. As all 4 reversible, terminator-bound dNTPs are persent during each sequencing cycle, natural competition minimizesincorporation bias and greatly reduces raw error rates compared to other technologies. The result is highly accurate base-by-base sequencing that virtually eliminates sequence-context-specificerrors, even within repetitive sequence regions and homopolymers. 49) Raw dataSequencing data is converted into raw data for the analysis.