Accurate Determination of Copy Number States for Multiallelic Copy Number Variations

Using next-generation sequencing (NGS) and Bio-Rad’s Droplet Digital PCR (ddPCR™) technology, researchers at Harvard Medical School and Bio-Rad’s Digital Biology Center have solved the technical challenge of accurately counting the diverse copy number states of multiallelic copy number variations (mCNVs).

“After a long period of not being able to do precise genetic analysis of mCNVs in human genetics, these tools — both whole-genome sequencing and nimble Droplet Digital PCR assays — will finally enable careful genetic analysis of mCNVs in human cohorts. Reassuringly, these approaches appear to yield results that agree strongly with each other,” said Steven McCarroll, professor of genetics at Harvard Medical School, director of genetics for the Stanley Center for Psychiatric Research at the Broad Institute, and senior author of the Nature Genetics paper in which these findings were published.

McCarroll’s team found that mCNVs are responsible for nearly 90% of the observed differences in gene copy number, or gene dosage, between humans.

“mCNVs are much more extensive and have a larger impact on gene-dosage variation than previously thought,” said McCarroll. “We also determined that mCNVs contribute substantially to gene expression variation, suggesting that they have the potential to contribute to variation in phenotypes.”
Because some genes affected by multicopy regions, such as HPR1 and ORM1, have disease associations, further investigation of mCNVs could enable studies of how copy number changes in these regions impact human phenotypes and disease, said Jennifer Berman, one of the paper’s coauthors and staff scientist at Bio-Rad’s Digital Biology Center.

Droplet Digital PCR Validates NGS Analysis

mCNVs have been notoriously difficult to study, due to a specific technical challenge: the inability to discriminate between higher order, consecutive copy number states (for example, six vs. seven) using existing low-precision techniques such as microarrays or standard qPCR.
The advent of NGS in the last decade, and ddPCR in the last few years, has broadened and deepened the ability of researchers to perform genetic analysis.

"The only two methods available to robustly call consecutive high copy number states are NGS with the new algorithms McCarroll’s lab developed as part of the study and Droplet Digital PCR,” said Berman.

Droplet Digital PCR, a technology developed by Bio-Rad that has been referenced in nearly 200 papers since it came to market in late 2011, is an ultraprecise and sensitive form of PCR that enables discrimination of small fold differences in target DNA copy numbers.
McCarroll’s research team used advanced computational methods in conjunction with existing NGS data to identify and catalog more than 8,500 CNVs in the human genome. In the analysis of 849 human genome sequences from the 1000 Genomes Project, approximately 3,900 duplication CNVs were discovered, of which roughly one-third were found to be mCNVs.

To validate their NGS computational approach, which uncovered diploid copy numbers ranging from one to 15, Dr. McCarroll and his team used Bio-Rad Laboratories’ award-winning QX200™ Droplet Digital PCR (ddPCR) System. Results obtained using the two techniques were highly consistent: per individual genome, the number of copies of mCNVs quantified by ddPCR had a 99% match rate with those of NGS. This type of orthogonal validation reinforces each technique’s approach and strengthens the conclusions of the study.