Project description:Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a procedure using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to sub-femtograms of DNA. The amplicons from 5 ng and 0.5 ng DNA, which were from originally good quality of gDNA (05-050), or partially degraded gDNA (04-018), faithfully demonstrated all previously known heterozygous segmental duplications and deletions (3 Mb to 18 kb) located on chromosome 22 and even a homozygous deletion smaller than 1 kb with high resolution chromosome-wide CGH. Specifically, HR-CGH with 5 ng-input gDNA-derived amplicon detected all previously known chromosomal segmental aberrations in chromosome 22 in samples from two different probands, and was indistinguishable from the HR-CGH result with native gDNA from the same probands (Fig. 4, Fig. S3 and S4). The break points were also precisely demonstrated. These include a heterozygous genomic segmental duplication (3 copies, 3 Mb in size, sample 05-050, Fig. 4) and 2 different heterozygous deletions (1 copy, 1.4 Mb and 18 kb respectively, sample 04-018, Fig. S4), all of which are located in or bounded by regions of low copy repeats (LCRs). In addition, a previous known homozygous deletion of 975 bp (in 04-018 and 05-050) was again accurately demonstrated (05-050 data showed in Fig. 4c), although sometimes (04-018) the data was a little noisier than with unamplified DNA (Fig. S4d). In contrast, the Wpa-40oC resulted in abundant signal noise and failed in detection of these copy number aberrations (Fig. 4, Fig. S3, S4). Impressively, HR-CGH with 0.5 ng gDNA-derived amplicons via Wpa also clearly detected the known CNVs, although noisier (Fig. S4). The 0.1ng gDNA derived amplicons via Wpa could not unambiguously show CNVs because of higher variability of signals, but the CNVs’ patterns were mostly well maintained (Fig. S4 for 04-018). We did also notice some locus-imbalance in the amplicon, however this was minimized, and was reproducible when the input was above a certain threshold amount, and it could be well compensated if the same amplified reference sample was applied in parallel as showed above. Keywords: Whole-pool amplification, high resolution comparative genome hybridization (HR-CGH)

Project description:Goal: To identify copy number variation in normal individuals using high density, non-polymorphic oligonucleotide probes Background DNA sequence diversity within the human genome may be more greatly affected by copy number variations (CNVs) than single nucleotide polymorphisms (SNPs). Although the importance of CNVs in genome wide association studies (GWAS) is becoming widely accepted, the optimal methods for identifying these variants are still under evaluation. We have previously reported a comprehensive view of CNVs in the HapMap DNA collection using high density 500K EA (Early Access) SNP genotyping arrays which revealed greater than 1,000 CNVs ranging in size from 1kb to over 3Mb. Although the arrays used most commonly for GWAS predominantly interrogate SNPs, CNV identification and detection does not necessarily require the use of DNA probes centered on polymorphic nucleotides and may even be hindered by the dependence on a successful SNP genotyping assay. Results In this study, we have designed and evaluated a high density array predicated on the use of non-polymorphic oligonucleotide probes for CNV detection. This approach effectively uncouples copy number detection from SNP genotyping and thus has the potential to significantly improve probe coverage for genome-wide CNV identification. This array, in conjunction with PCR-based, complexity-reduced DNA target, queries over 1.3M independent NspI restriction enzyme fragments in the 200bp to 1100bp size range, which is a several fold increase in marker density as compared to the 500K EA array. In addition, a novel algorithm was developed and validated to extract CNV regions and boundaries. Conclusions Using a well-characterized pair of DNA samples, close to 200 CNVs were identified, of which nearly 50% appear novel yet were independently validated using quantitative PCR. The results indicate that non-polymorphic probes provide a robust approach for CNV identification, and the increasing precision of CNV boundary delineation should allow a more complete analysis of their genomic organization. A set of five genomic DNA samples containing different numbers of X chromosomes (1X to 5X sample set, including NA15510 and NA10851) were hybridized to Nsp copy number (CN) arrays in triplicate to evaluate detection of copy number variation using high density, non-polymorphic oligonucleotide probes. 6 Hapmap samples were hybridized to Nsp CN arrays to evaluate Mendelian inheritance of CNVs.

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Homo sapiens

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