Research Area:	Genetics and Genomics
Keywords:	Genome, DNA sequencing and Single Molecule
Web Links:	Wellcome Trust website

Despite recent advances, the current generation of sequencing methods are still too expensive, and time-consuming for genome sequencing of individuals to be routinely implemented on a large scale. Moreover the available technologies provide relatively short reads with no long-range genomic context. Assembly requires computationally intensive comparisons to a reference genome and unless complex and expensive mate-paired library preparations are used, little information on haplotypes or structural variation is obtained. The Mir group is developing new methods to address whole genomes or to pull out specific parts of genomes, to determine DNA base sequence and to pinpoint the sequence reads to their actual location within an individual genome which should enable de novo and relatively gap-free assembly of genomes.

Name	Department	Institution	Country
Jonas Tegenfeldt		Lund University	Sweden
Dr Ioannis Ragoussis	Wellcome Trust Centre for Human Genetics	Oxford University	UK
Achillefs Kapanides		Oxford University	UK
Dr Emanuela V Volpi	Wellcome Trust Centre for Human Genetics	Oxford University	UK
Anders Kristensen		Danish Technical university	DTU
Tom Brown		University of Southampton	UK
Hagan Bayley FRS	Chemistry	Oxford	UK

Mir KU. 2009. Sequencing genomes: from individuals to populations. Brief Funct Genomic Proteomic, 8 (5), pp. 367-378. Read abstract | Read more

The whole genome sequences of Jim Watson and Craig Venter are early examples of personalized genomics, which promises to change how we approach healthcare in the future. Before personal sequencing can have practical medical benefits, however, and before it should be advocated for implementation at the population-scale, there needs to be a better understanding of which genetic variants influence which traits and how their effects are modified by epigenetic factors. Nonetheless, for forging links between DNA sequence and phenotype, efforts to sequence the genomes of individuals need to continue; this includes sequencing sub-populations for association studies which analyse the difference in sequence between disease affected and unaffected individuals. Such studies can only be applied on a large enough scale to be effective if the massive strides in sequencing technology that have recently occurred also continue. Hide abstract

Mir KU, Qi H, Salata O, Scozzafava G. 2009. Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template. Nucleic Acids Res, 37 (1), pp. e5. Read abstract | Read more

Next generation sequencing methods that can be applied to both the resequencing of whole genomes and to the selective resequencing of specific parts of genomes are needed. We describe (i) a massively scalable biochemistry, Cyclical Ligation and Cleavage (CycLiC) for contiguous base sequencing and (ii) apply it directly to a template captured on a microarray. CycLiC uses four color-coded DNA/RNA chimeric oligonucleotide libraries (OL) to extend a primer, a base at a time, along a template. The cycles comprise the steps: (i) ligation of OLs, (ii) identification of extended base by label detection, and (iii) cleavage to remove label/terminator and undetermined bases. For proof-of-principle, we show that the method conforms to design and that we can read contiguous bases of sequence correctly from a template captured by hybridization from solution to a microarray probe. The method is amenable to massive scale-up, miniaturization and automation. Implementation on a microarray format offers the potential for both selection and sequencing of a large number of genomic regions on a single platform. Because the method uses commonly available reagents it can be developed further by a community of users. Hide abstract

Mir KU. 2006. Ultrasensitive RNA profiling: counting single molecules on microarrays. Genome Res, 16 (10), pp. 1195-1197. | Read more

Mir KU, Southern EM. 2000. Sequence variation in genes and genomic DNA: methods for large-scale analysis. Annu Rev Genomics Hum Genet, 1 (2000), pp. 329-360. Read abstract | Read more

The large-scale typing of sequence variation in genes and genomic DNA presents new challenges for which it is not clear that current technologies are sufficiently sensitive, robust, or scalable. This review surveys the current platform technologies: separation-based approaches, which include mass spectrometry; homogeneous assays; and solid-phase/array-based assays. We assess techniques for discovering and typing variation on a large scale, especially that of single-nucleotide polymorphisms. The in-depth focus is the DNA chip/array platform, and some of the published large-scale studies are closely examined. The problem of large-scale amplification is addressed, and emerging technologies for present and future needs are indicated. Hide abstract

Mir KU, Southern EM. 1999. Determining the influence of structure on hybridization using oligonucleotide arrays. Nat Biotechnol, 17 (8), pp. 788-792. Read abstract | Read more

We have studied the effects of structure on nucleic acid heteroduplex formation by analyzing hybridization of tRNAphe to a complete set of complementary oligonucleotides, ranging from single nucleotides to dodecanucleotides. The analysis points to features in tRNA that determine heteroduplex yield. All heteroduplexes that give high yield include both double-stranded stems as well as single-stranded regions. Bases in the single-stranded regions are stacked onto the stems, and heteroduplexes terminate at potential interfaces for coaxial stacking. Heteroduplex formation is disfavored by sharp turns or a lack of helical order in single-stranded regions, competition from bases displaced from a stem, and stable tertiary interactions. The study is relevant to duplex formation on oligonucleotide microarrays and to antisense technologies. Hide abstract

Southern E, Mir K, Shchepinov M. 1999. Molecular interactions on microarrays. Nat Genet, 21 (1 Suppl), pp. 5-9. Read abstract | Read more

The structural features of nucleic acid probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implication for the hybridization process. We discuss how arrays of oligonucleotides provide powerful tools to study the molecular basis of these interactions on a scale which is impossible using conventional analysis. Hide abstract