Illumina sequencing is one of the new generation techniques of DNA sequencing. It was developed by researchers at Manteia Predictive Medicine which was acquired by Solexa in 2004 and Solexa, was later acquired by Illumina. Hence the name, Illumina sequencing. This sequencing method is based on using reversible dye-terminators that enable the identification of single bases as they are introduced into DNA strands. The use of clonal arrays and massively parallel sequencing of short reads using reversible terminators was subsequently referred to as sequencing by synthesis technology or SBS.
This method is often used to sequence difficult regions, such as homopolymers and repetitive sequences. It can also be used for whole-genome sequencing, region targeted sequencing, de novo sequencing transcriptome analysis, small RNA analysis, methylation profiling, and genome-wide protein-nucleic acid interaction analysis.
DNA molecules are first attached to primers on a slide and the clones are amplified by bridge amplification so that local colonies of DNA or “DNA clusters” are formed. This cluster generation technology had been invented and developed by Dr Pascal Mayer and Dr Laurent Farinelli in 1996 at Glaxo-Welcome’s Geneva Biomedical Research Institute (GBRI).
The DNA templates are sequenced base by base, in parallel, using four types (adenine, cytosine, guanine, and thymine) of reversible terminate bases (RT bases). These bases are fluorescently labeled with a different color and attached with a terminal 3’ blocking agent. The four bases then compete for binding sites on the template DNA to be sequenced and unbound nucleotides are washed away. This natural competition ensures the highest accuracy. After each synthesis a laser is used to excite the clusters resulting in the removal of the 3’ terminal blocking group and the probe. The fluorescent color specific to one of the four bases is then visible, allowing for sequence identification and the beginning of the next cycle. The process is repeated until the full DNA molecule is sequenced. This technique allows for very large arrays of DNA colonies to be captured by sequential images taken from a single camera.
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 like Pyrosequencing.
Illumina sequencing has been used to research transcriptomes of the sweet potato and the gymnosperm genus Taxus and research in oncology. Illumina unveiled its HiSeq X (pronounced “High Seek 10”) on January 14th, 2014. This is the world’s first DNA-crunching supercomputer designed to process 20,000 genomes per year at a cost of $1,000 each. Previously it cost about $10,000 to sequence a human genome.
admin January 17th, 2014
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