The following are some of the research goals that the Mir group is focused on:
There is a general need for technology that can selectively resequence specific parts of genomes. For example, Genome Wide Association Studies (GWAS) point to regions of human genomes linked to disease susceptibility, but directed resequencing of these regions is needed to pinpoint causative mutations. Current or so-called "next" generation technologies are "shotgun" resequencing methods that are not intrinsically capable of sequencing selected subsets of the genome. Therefore, presently, selection is performed separately before entering the sequencing pipeline. The selection methods that have been described so far are relatively laborious and are not entirely satisfactory in the coverage they obtain over the selected region(s). The Mir group is developing a streamlined approach for selective sequencing that fully integrates selection with sequencing (Mir et al, NAR). The workflow comprises microarray design, selection by microarray capture, a ligation-based "second generation" sequencing biochemistry that extends from the capture probe to read sequence in a template-directed reaction, and bioinformatics for base-calling and quality scoring. We are working with collaborators within the WTCHG to apply the technology to clinical-relevant targets.
Sequencing in a long-range context
In the current generation of technologies, sequencing reads have become shorter than those provided by the traditional "Sanger" method. This approach is at odds with the growing view that long-range structural variation (SV) in the genome is at least as important as localised sequence variation in determining phenotypes in biology and disease. Although chromosomes each comprise a single linear DNA molecule, genomic DNA becomes sheared and randomized during extraction. Our hypothesis is that base sequence can be reconciled with long-range genome structure by obtaining sequence directly on long lengths of single genomic DNA molecules in their native linear state. We have developed a platform for displaying long lengths of single genomic DNA on a surface using a microarray; we are also investigating as part of the READNA EU consortium, novel fluidic devices that stretch DNA by nano-confinement. These methods will provide a "bird's eye" view of the genome upon which we will map the location of sequence reads in their long-range context. This will allow provide the diploid sequence of the human genome and enable haplotypes and all forms of structural variation to be identified; we are also working towards mapping sites methylation upon the genomic DNA. The technology has the potential to lead to new understanding of how the sequence, structure and epigenetic regulation of the genome combine to affect phenotype.
Real Time DNA Sequencing
The cyclical nature of second generation sequencing biochemistries limit the read-lengths they can be provided within a reasonable time-frame and cost. Recently a "real-time" DNA sequencing approach has been described which tracks a template-directed DNA synthesis reaction on single molecules without the need for reagent exchange cycles thus potentially saving on time and cost (see Eid et al, Science, 323:133-138; 2009). The Mir group believes that real-time sequencing is important if we are to obtain read-lengths that span lengths of the majority of structural variants in the genome. In collaboration with partners in the READNA consortium and elsewhere, we are developing novel real-time DNA sequencing biochemistries based on Fluorescence Resonance Energy Transfer (FRET) and other optical methods.
We are working with collaborators within the READNA consortium to develop methods for genome analysis using nano-scale pores through which single genomic DNA molecules can traverse.
Mir et al. 2009 Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template, Nucleic Acids Res. Jan;37(1):e5. Epub 2008 Nov 16. PMID: 19015154