HarvardPGP54K
Significantly improved multiplex padlock capturing and large scale sequencing reveal hypermutable CpG variations
53,777 padlock probes were designed to cover all CpGs in non-repetitive  regions of chromosome 21.  Sequencing libraries were constructed for six  subjects (one replicated, so seven libraries) and sequenced twice, once on  an Illumina Genome Analyzer 1 (run 1), and once on an Illumina Genome  Analyzer 2 (run 2). For run 1, 41 base reads were obtained for each  library, and for run 2, 36 base reads were obtained; in either case, only  the first 36 bases were used to map reads.  In each 36 base read, the  first 25 bases corresponded to the ligation arm of the padlock probe and  were used to map the read; these bases copy synthesized DNA in the padlock  probes and therefore do not provide copies of subjects' genomic DNA.   Actual subject genomic DNA occupies read positions 26-35 (10 bases).  The  two ends of the padlock probe -- the ligation arm and the extension arm --  hybridize to genomic DNA and were designed so that the gap between them  was 40bp (only 10 of which were sequenced).  Probes were designed so that  the two arms could only hybridize and form a product of the correct size  in one location in the genome.  Thus, even though the 36 base reads may in  some cases align to multiple genome locations, the presence of these  sequences in the sequencing library ensures that they were obtained from  the targeted location.
To take full advantage of the power of next generation sequencing requires  large scale multiplex capturing and amplification of genomic regions of  interest in multiple samples.  We improved a previously developed padlock  probe-based approach by 10,000-fold and evaluated it by identifying  genetic variations in hypermutable CpG rich regions, tiling CpGs across  human chromosome 21 with 53,777 probes in an unbiased manner. Two to three  million mapped sequences derived from a single Illumina Genome Analyzer  lane enable observation of 90.8%-94.0% of target sites with at least one  read. Although the sites were not uniformly captured, ~85% and ~55% of all  targets fell within a 100- and 10-fold range of each other, respectively .  A total of 442937 genotypes were computed with confidence across six  subjects and examined for single nucleotide polymorphisms and were found  to be in between 98.4% and 100% agreement with independently obtained  genotype data. We detected as many as 502 sites of variation not present  in dbSNP, including 362 in targeted CpG locations not among the 2640 in  dbSNP.  CpG->CpA and CpG->TpG variations were found to be ~12.5x more  abundant than non-CpG variations.  Variation rates differed among subjects  in a possibly ancestry-related manner.  Our success suggests that  genotyping hypermutable CpGs may be an efficient way of identifying common  and rare mutations in human diseases and that padlock-probe based  sequencing can reveal important aspects of human variation generally. We  identified areas for further improvement and study.