Illumina dye sequencing

Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris. It was developed by Shankar Balasubramanian and David Klenerman of Cambridge University, 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 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. Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris. It was developed by Shankar Balasubramanian and David Klenerman of Cambridge University, 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 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. Illumina sequencing technology works in three basic steps: amplify, sequence, and analyze. The process begins with purified DNA. The DNA gets chopped up into smaller pieces and given adapters, indices, and other kinds of molecular modifications that act as reference points during amplification, sequencing, and analysis. The modified DNA is loaded onto a specialized chip where amplification and sequencing will take place. Along the bottom of the chip are hundreds of thousands of oligonucleotides (short, synthetic pieces of DNA). They are anchored to the chip and able to grab DNA fragments that have complementary sequences. Once the fragments have attached, a phase called cluster generation begins. This step makes about a thousand copies of each fragment of DNA. Next, primers and modified nucleotides enter the chip. These nucleotides have reversible 3' blockers that force the polymerase to add on only one nucleotide at a time as well as fluorescent tags. After each round of synthesis, a camera takes a picture of the chip. A computer determines what base was added by the wavelength of the fluorescent tag and records it for every spot on the chip. After each round, non-incorporated molecules are washed away. 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 continues until the full DNA molecule is sequenced. With this technology, thousands of places throughout the genome are sequenced at once via massive parallel sequencing. The first step after DNA purification is tagmentation. Transposases randomly cut the DNA into short segments ('tags'). Adapters are added on either side of the cut points (ligation). Strands that fail to have adapters ligated are washed away. The next step is called reduced cycle amplification. During this step, sequences for primer binding, indices, and terminal sequences are added. Indices are usually six base pairs long and are used during DNA sequence analysis to identify samples. Indices allow for up to 96 different samples to be run together. During analysis, the computer will group all reads with the same index together. The terminal sequences are used for attaching the DNA strand to the flow cell.Illumina uses a 'sequence by synthesis' approach. This process takes place inside of an acrylamide-coated glass flow cell. The flow cell has oligonucleotides (short nucleotide sequences) coating the bottom of the cell, and they serve to hold the DNA strands in place during sequencing. The oligos match the two kinds of terminal sequences added to the DNA during reduced cycle amplification. As the DNA enters the flow cell, one of the adapters attaches to a complementary oligo.