Many diseases, including cancer, involve CpG mutations. Cost-effective sequencing can be achieved using multiplexed targeted amplification. Here padlock capturing technology efficiency, sensitivity, and uniformity was vastly improved. The authors designed padlock probes covering 24% of CpGs on chromosome 21. They sequenced libraries on the Genome Analyzer II. Efficiency in the capture of 55,000 exons was increased 10,000 fold by extending the genomic DNA/padlock probe hybridization reaction time, increasing amounts of padlock probes, and optimization of dNTP concentrations.

Genes residing in the chromosomal region suspected to be linked with the neurological disorder sensory/motor neuropathy with ataxia (SMNA) were investigated. Using massively parallel sequencing on the Genome Analyzer, and other methods of analysis, they found a nonsynonymous variant in the interferon-related developmental regulator gene 1 (IFRD1) in the suspect region (7q22-q32), which was not found in healthy controls. This gene is known to be localized to brain and spinal cord areas commonly affected in SNMA.

The authors adapted a molecular inversion probe (MIP) protocol to enable “simultaneous amplification and accurate shotgun sequencing of 50,000 exons,” using the Genome Analyzer. Of these targets, 91% were captured, compared to 18% previously. They also examined a method to directly sequence MIP amplicons and found a greater proportion of mapped reads compared to shotgun sequencing (90.4% vs 56.8%), with almost 100% specificity. This eliminated the requirement for shotgun library construction, and only needed submicrogram amounts of DNA.