Illumina dye sequencing

Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. It was based on inventions of S Balasubramanian and D Klenerman of Cambridge University,[1] who subsequently founded Solexa, a company later acquired by Illumina. This sequencing method is based on reversible dye-terminators that enable the identification of single bases as they are introduced into DNA strands. It is often employed to sequence difficult regions, such as homopolymers and repetitive sequences. It can also be used for whole-genome and region sequencing, transcriptome analysis, metagenomics, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis.[2][3]

Procedure

Illumina dye sequencing begins with the attachment of DNA molecules to primers on a slide, followed by amplification of that DNA to produce local colonies. This "DNA cluster" generation technology had been invented and developed in late 1996 at Glaxo-Welcome's Geneva Biomedical Research Institute (GBRI), by Dr Pascal Mayer and Dr Laurent Farinelli,[4] and was publicly presented for the first time in 1998.[5] Solexa's proprietary chemistry, which also required an engineered polymerase, was developed at its site in Gt. Chesterford near Cambridge (UK), the initial commercial versions of the clusters technology were also perfected here. The four types (adenine, cytosine, guanine, and thymine) of reversible terminate bases are added, each fluorescently labeled with a different color and attached with a blocking group. The four bases then compete for binding sites on the template DNA to be sequenced and non-incorporated molecules are washed away. After each synthesis, a laser is used to excite the dyes and a photograph of the incorporated base is taken. A chemical deblocking step is then used in the removal of the 3’ terminal blocking group and the dye in a single step. The process is repeated until the full DNA molecule is sequenced.[3]

Comparison with other sequencing methods

This technique offers a number of advantages over traditional sequencing methods such as Sanger sequencing. Due to the automated nature of Illumina dye sequencing it is possible to sequence multiple strands at once and gain actual sequencing data quickly. Additionally, this method only uses DNA polymerase as opposed to multiple, expensive enzymes required by other sequencing techniques (i.e. pyrosequencing).[6]

Examples of use

Illumina sequencing has been used to research transcriptomes of the sweet potato[7] and the gymnosperm genus Taxus.[8]

References

  1. ↑ http://technology.illumina.com/technology/next-generation-sequencing/solexa-technology.html
  2. ↑ Illumina - Sequencing Technology
  3. ↑ 3.0 3.1 Meyer M., Kircher M.(2010).Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing. Cold Springs Harbor Protocols".doi:10.1101/pdb.prot5448.
  4. ↑ patents WO 9844151, WO 9844152
  5. ↑ DNA colony massively parallel sequencing ams98 presentation
  6. ↑ Pettersson E., Lundeberge J., Ahmadian A.(2008).Generation of sequencing technologies. Genomics".pp. 105-111.
  7. ↑ Wang, Z; Fang, B; Chen, J; Zhang, X; Luo, Z; Huang, L; Chen, X; Li, Y (Dec 24, 2010). "De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas).". BMC Genomics 11: 726. doi:10.1186/1471-2164-11-726. PMC 3016421. PMID 21182800.
  8. ↑ Hao, Da Cheng; Ge, GuangBo; Xiao, PeiGen; Zhang, YanYan; Yang, Ling; Ellegren, Hans (22 June 2011). "The First Insight into the Tissue Specific Taxus Transcriptome via Illumina Second Generation Sequencing". PLoS ONE 6 (6): e21220. doi:10.1371/journal.pone.0021220.