Ions in Barrett’s Esophagus [113,114] and CPI-455 biological activity ulcerative colitis [115], among a variety of other cancer-predisposing diseases. Navin et al recently used the profile of CGH-identified copy number changes to study the clonal architecture of different regions of advanced breast cancers through phylogenetic inference [116]. Direct genomic sequencing provides the most detailed means possible of identifying clonal mutant markers. In contrast to conventional capillary-based techniques where individual PCR products or bacterial clones must be sequenced individually, a powerful new class of “Next Generation” sequencing technologies allows for simultaneous genotyping of tens of billions of base pairs [117]. The rapidly decreasing costs associated with these platforms have recently made it feasible to sequence the entire aggregate genome of a tissue sample without any regional targeting. From a clone detection perspective this means that multiple types of mutations of all functional varieties (both likely passengers and suspected drivers) can be simultaneously assessed. While it only takes a single clonal mutation to identify an expanded population, the redundancy conferred by screening the entire genome provides a huge amount of additional lineage data with the potential to be used for subanalyses such as approximation of a clone’s mitotic age or the phylogenetic relationship between different clones. The digital manner in which these novel sequencing technologies operate lend them a much greater dynamic range of sensitivity than conventional techniques, making it possible to resolve populations that are subclonal relative to a collected sample. Such an ability means that in situations where spatial coherence of an expanding clone is not maintained, for example in myelodysplasia preceding blood cancers, detection at an early stage can still be accomplished [118]. Similarly, a tolerance for clone mixing should allow for convenient, minimally invasive sampling techniques that disrupt cohesive growth patterns in epithelial tissues such as cell isolation from lavage, scrapings or body fluids rather than biopsy. The relatively high error rate of individual sequencing reads currently limits the average depth to which rare subclonal mutations can be accurately detected to about 2 orders of magnitude below pure clonality [26]. A variety of improvements at the level of chemistry, hardware and analysis are continuing to enhance this resolution for all current platforms [118,119]. An even newer generation of exotic “Fourth Generation” sequencing technologies on the horizon promises ultra-long read lengths with the ability to continuously re-sequence theActidione biological activity NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSemin Cancer Biol. Author manuscript; available in PMC 2011 October 15.Salk and HorwitzPagesame molecule for extremely accurate detection of rare molecular populations [120?22]. The pace of innovation in this area is staggering. Perhaps the only thing that can be stated with certainty about the technologies that will be available five years in the future is that they will look nothing like those from five years in the past.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript9. The complex interpretation of clonality: false negatives, positives and assumptionsThe reasoning associated with clonality determination is complex and many potential confounders exist. In this section we highlight seven important ideas for consi.Ions in Barrett’s Esophagus [113,114] and ulcerative colitis [115], among a variety of other cancer-predisposing diseases. Navin et al recently used the profile of CGH-identified copy number changes to study the clonal architecture of different regions of advanced breast cancers through phylogenetic inference [116]. Direct genomic sequencing provides the most detailed means possible of identifying clonal mutant markers. In contrast to conventional capillary-based techniques where individual PCR products or bacterial clones must be sequenced individually, a powerful new class of “Next Generation” sequencing technologies allows for simultaneous genotyping of tens of billions of base pairs [117]. The rapidly decreasing costs associated with these platforms have recently made it feasible to sequence the entire aggregate genome of a tissue sample without any regional targeting. From a clone detection perspective this means that multiple types of mutations of all functional varieties (both likely passengers and suspected drivers) can be simultaneously assessed. While it only takes a single clonal mutation to identify an expanded population, the redundancy conferred by screening the entire genome provides a huge amount of additional lineage data with the potential to be used for subanalyses such as approximation of a clone’s mitotic age or the phylogenetic relationship between different clones. The digital manner in which these novel sequencing technologies operate lend them a much greater dynamic range of sensitivity than conventional techniques, making it possible to resolve populations that are subclonal relative to a collected sample. Such an ability means that in situations where spatial coherence of an expanding clone is not maintained, for example in myelodysplasia preceding blood cancers, detection at an early stage can still be accomplished [118]. Similarly, a tolerance for clone mixing should allow for convenient, minimally invasive sampling techniques that disrupt cohesive growth patterns in epithelial tissues such as cell isolation from lavage, scrapings or body fluids rather than biopsy. The relatively high error rate of individual sequencing reads currently limits the average depth to which rare subclonal mutations can be accurately detected to about 2 orders of magnitude below pure clonality [26]. A variety of improvements at the level of chemistry, hardware and analysis are continuing to enhance this resolution for all current platforms [118,119]. An even newer generation of exotic “Fourth Generation” sequencing technologies on the horizon promises ultra-long read lengths with the ability to continuously re-sequence theNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSemin Cancer Biol. Author manuscript; available in PMC 2011 October 15.Salk and HorwitzPagesame molecule for extremely accurate detection of rare molecular populations [120?22]. The pace of innovation in this area is staggering. Perhaps the only thing that can be stated with certainty about the technologies that will be available five years in the future is that they will look nothing like those from five years in the past.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript9. The complex interpretation of clonality: false negatives, positives and assumptionsThe reasoning associated with clonality determination is complex and many potential confounders exist. In this section we highlight seven important ideas for consi.
