This process involves a mixture of techniques: bacterial cloning or PCR; template purification; labelling of DNA fragments using the chain termination method with energy transfer, dye-labelled dideoxynucleotides and a DNA polymerase; capillary electrophoresis; and fluorescence detection that provides four-colour plots to reveal the DNA sequence.

Finished grade A quality measure for a sequenced genome. A finished-grade genome, commonly referred to as a ‘finished genome’, is of higher quality than a draft-grade genome, with more base coverage and fewer errors and gaps (for example, the human genome reference contains 2.85 Gb, covers 99% of the genome with 341 gaps, and has an error rate of 1 in every 100,000 bp).

This recombinant DNA molecule is made up of a known region, usually a vector or adaptor sequence to which a  universal primer can bind, and the target sequence, which is typically an unknown portion to be sequenced.

Assays that use next-generation sequencing technologies. They include methods for determining the sequence content and abundance of mRNAs, non-coding RNAs and small RNAs (collectively called RNA–seq) and methods for measuring genome-wide profiles of immunoprecipitated DNA–protein complexes (ChIP–seq), methylation sites (methyl–seq) and DNase I hypersensitivity sites (DNase–seq).

A fragment library is prepared by randomly shearing genomic DNA into small sizes of <1kb, and requires less DNA  than would be needed for a mate-pair library.

A genomic library is prepared by circularizing sheared DNA that has been selected for a given size, such as 2 kb, therefore bringing the ends that were previously distant from one another into close proximity. Cutting these circles into linear DNA fragments creates mate-pair templates. 

A nucleotide or probe that does not contain a fluorescent label. It can be generated from its cleavage and carry-over from the previous cycle or be hydrolysed in situ from its dye-labelled counterpart in the current cycle.

A total internal reflection fluorescence imaging device produces an evanescent wave — that is, a near-field stationary excitation wave with an intensity that decreases exponentially away from the surface. This wave propagates across a boundary surface, such as a glass slide, resulting in the excitation of fluorescent molecules near (<200 nm) or at the surface and the subsequent collection of their emission signals by a detector.

Large numbers of genetically engineered DNA polymerases can be created by either site-directed or random mutagenesis, which leads to one or more amino acid substitutions, insertions and/or deletions in the polymerase. The goal of this approach is to incorporate modified nucleotides more efficiently during the sequencing reaction.

These are only useful for single-molecule techniques and are produced by sequencing the same template molecule more than once. The data are then aligned to produce a ‘consensus read’, reducing stochastic errors that may occur  in a given sequence read. 

An oligonucleotide sequence in which one interrogation base is associated with a particular dye (for example, A in the first position corresponds to a green dye). An example of a one-base degenerate probe set is ‘1-probes’, which indicates that the first nucleotide is the interrogation base. The remaining bases consist of either degenerate (four  possible bases) or universal bases. 

An oligonucleotide sequence in which two interrogation bases are associated with a particular dye (for example, AA, CC, GG and TT are coded with a blue dye). ‘1,2-probes’ indicates that the first and second nucleotides are the interrogation bases. The remaining bases consist of either degenerate or universal bases.

A nucleotide substitution will have two colour calls, one from the 5′ position and one from the 3′ position of the dinucleotide sequence. When compared with a reference genome, base substitution in the target sequence is encoded by two specific, adjacent colours. In figure 3b, the sequence ‘CCT’ is encoded as blue-yellow (‘CC’ = blue; ‘CT’ = yellow), but substituting the middle ‘C’ for ‘A’ would result in two colour changes to green-red. Any other colour sequence can be discarded as an error. 

With two-base-encoded probes, the fluorescent signal or colour obtained during imaging is associated with four dinucleotide sequences having a 5′- and 3′-base. Colour space is the sequence of overlapping dinucleotides that codes four simultaneous nucleotide sequences. Alignment with a reference genome is the most accurate method for  translating colour space into a single nucleotide sequence. 

This nanostructure device is 100 nm in diameter, which is smaller than the 532 nm and 643 nm laser wavelengths used in the Pacific Biosciences platform. Light cannot propagate through these small waveguides, hence the term zero-mode. These aluminium-clad waveguides are designed to produce an evanescent wave (see the ‘total internal reflection fluorescence’ glossary term) that substantially reduces the observation volume at the surface of the polymerase reaction down to the zeptolitre range (10–21 l). This provides an advantage for the polymerization  reaction, which can be performed at higher dye-labelled nucleotide concentrations.

This is generally a system that consists of two fluorescent dyes, one being a donor dye (a bluer fluorophore) and the  other an acceptor dye (a redder fluorophore). When the two dye molecules are brought into close proximity (usually ≤30 nm), the energy from the excited donor dye is transferred to the acceptor dye, increasing its emission intensity signal.

All sequence variants other than single-nucleotide variants, including block substitutions, insertions or deletions, inversions, segmental duplications and copy-number differences. 

A project aimed at discovering rare sequence variants with minor allele frequencies of 1% in normal genomes derived

from HapMap samples

A project aimed at developing and validating cost-effective, high-throughput technologies for resequencing all of the

protein-coding regions of the human genome.

A project aimed at discovering single-nucleotide variants and structural variants that are associated with major cancers, such as brain cancer (glioblastoma multiforme), lung cancer (squamous carcinoma) and ovarian cancer (serous cystadenocarcinoma). 

A project aimed at providing open access to human genome sequences from volunteers and to develop tools for interpreting this information and correlating it with related personal medical information.

 assessing the combined contribution of multiple genetic variants acting within biological pathways provides biological insight into disease susceptibility 

Type 1Diabetes, Crohn’s Disease, and Rheumatoid Arthritis. 

All transcribed mRNA 

in a particular tissue at a particular time

northern blot

PCR (polymerase chain reaction)

microarrays

next generation sequencing

“spotted” cDNA on arrays by robot

Quality of spots may vary across arrays. 200-1000 bp. two color

20-80 nt

Quality of spots is generally more constant across arrays. single color

Two-color laser scanning allows quantitative studies of gene-expression in two samples simultaneously

determine the relative abundance of transcripts 

annotate coding SNPs 

discover transcript isoforms 

identify regulatory RNAs 

characterize splice junctions 

Problem: multiple testing, finding significant differences by chance. So many false positives

Inflammation-mediated  

Immune-independent

Monocytes are required

Increased Interferonβ activity