Applications for the Illumina HiSeq 2000 Technology
DNA sequencing
Whole genome sequencing
Sequencing the complete DNA sequence of an organism is the first step in comparative genomics in order to identify conserved sequence regions between different species or phenotypically relevant differences. Resequenced genomes can be mapped on reference sequences in order to detect single base mutations, mutation hot spots, copy number variations, as well as inserted or deleted sequences. Therefore, resequencing builds the basis of sequence based karyotyping.
Targeted Resequencing
Targeted Resequencing reduces costs per sample and therefore is more feasible if only certain genomic regions ("targets") are of interest. Selected regions of the genome, e.g. the whole exome, are isolated, enriched and sequenced. This approach enables to take a closer look at specific regions to discover causal variants for a range of complex human diseases.
De-novo Sequencing
De-novo Sequencing is the initial sequencing of new, previously unknown genomic regions or whole genomes. Assembly of the resulting sequences can lead to the creation of reference genomes for novel species. It also gives the possibility to detect extensive rearrangements, e.g. in cancer genomes, or other biological relevant disorders.
ChIP-Sequencing
ChIP-Sequencing (or ChIP-Seq) is a technology which combines chromatin immunoprecipitation (ChIP) assays with massively parallel DNA sequencing. ChIP is a procedure to determine whether a given protein binds to a specific DNA sequence. First DNA and DNA-binding proteins are cross-linked, the cells are lysed and the DNA-bound proteins of interest are precipitated with specific antibodies. These antibodies are used to isolate the DNA of interest. The cross-linking is reversed and the protein digested. The remaining DNA fragments are amplified and sequenced. Therefore, the ChIP-Seq technology enables the analysis of DNA-protein interactions through the identification of potential binding sites of DNA-associated proteins and is a cost-effective method for the generation of precise maps of global binding sites for any protein of interest.
mRNA-Sequencing
mRNA-Seq is the method of choice for performing an efficient analysis of the whole transcriptome with respect to expression levels and RNA splice isoforms. After reverse transcription of the RNA the obtained cDNA is sequenced in order to get information about the composition of a RNA sample.
In addition to the identification of known gene transcripts and their splicing variants, it is possible to detect unknown genes and their location within the genome.
Bisulfite sequencing
Bisulfite sequencing uses bisulfite treatment of DNA to determine its pattern of methylation. DNA methylation is the most studied epigenetic mark. It predominantly involves the addition of a methyl-group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity.
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single-nucleotide resolution information about the methylation status of a segment of DNA. For bisulfite sequencing the fragmented DNA is first ligated to methylated adaptors, followed by a bisulfite treatment and a size fractionation. The resulting bisulfite treated DNA-library is then amplified and sequenced. Bisulfite sequencing allows the determination of methylation patterns at a genome-wide scale.
Small RNA sequencing
Small RNAs often have essential regulatory functions; e. g. microRNA (miRNA) is able to down-regulate gene expression by binding to RNA whereas silencing RNA (siRNA) interacts in the RNA interference (RNAi) pathway and prevents the integration of viruses and transposons into the host genome. The Illumina Genome Analyzer with its short read output is the system of choice for sequencing small RNAs. It performs the sequencing via a reverse transcription approach.
