AAT Bioquest

What is the general procedure of Illumina sequencing?

Posted July 22, 2020


Illumina sequencing is currently the most popular NGS platform on market, which consists of four basic steps: library preparation, cluster generation, sequencing, and data analysis.

  • Library preparation: The DNA or cDNA samples are randomly fragmented into ~500 bp sequences by either sonification or tagmentation. Right and left adapters along with the index sequences are then attached to these fragments, constructing a DNA library to be amplified and sequenced. Fragments that fail to have adapters ligated are washed away.
  • Cluster generation: The library of adapter-ligated fragments is loaded onto a flow cell, which is coated with oligoes complementary to the adapter. Fragments anchor on the cell surface via hybridization with complementary strands, and then are copied by bridge amplification PCR. Thousand copies of each original sequence are made, forming a distinct “clonal cluster” which enables production of sufficient signal during imaging process.
  • Sequencing: Primers and modified nucleotides are added onto the flow cell, and a strand complementary to the template is replicated by DNA polymerase. These nucleotides have a reversible blocking group so that the DNA polymerase can only add one nucleotide at a time onto the growing strand. After each round of synthesis, an image will be taken for the chip, and the newly added nucleotide can be identified by its fluorescent tag. Subsequently, the reversible blocking group and the fluorescent tag are removed from the nucleotide structure, allowing the addition of new nucleotide in the next cycles until the full DNA molecule is sequenced.
  • Analysis: Sequences are separated based on the unique indices added during the library preparation. Forward and reverse reads are matched to result in contiguous sequences. These contiguous sequences are then compared to the reference sequence, if any, for species/variation identification.
Additional resources

Helixyte™ Green *10,000X Aqueous PCR Solution*

6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*

Krishna, B. M., Khan, M. A., & Khan, S. T. (2019). Next-Generation Sequencing (NGS) Platforms: An Exciting Era of Genome Sequence Analysis. In Microbial Genomics in Sustainable Agroecosystems (pp. 89-109). Springer, Singapore.

Ju, J., Kim, D. H., Bi, L., Meng, Q., Bai, X., Li, Z., ... & Edwards, J. R. (2006). Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. Proceedings of the National Academy of Sciences, 103(52), 19635-19640.