Nextera™ sample prep enables breakthrough study of copy-number variation

The study of copy-number variation (CNV) in humans has contributed to our understanding of genetic uniqueness, as well as disease. Until recently, it was difficult to assess the number of repeated DNA sequences in the genome. In a recent publication, researchers at the 1000 Genomes Project and collaborators have invented new methods to study and find repetitive DNA sequences in the human genome, and have found that CNVs occur in only 7%-9% of human genes. They used the new techniques to compare the entire genomes of 159 individuals and were able to accurately assay previously unidentified duplicated genes.

For sequencing, the researchers picked and cultured 144 fosmid clones (from libraries prepared by shotgun cloning of genomic DNA) from eight selected individuals. After fosmid DNA purification, clone DNA was arrayed in a 96-well plate (two clones combined from unrelated loci for some). Bar-coded sequencing libraries were created separately from each well using the Nextera DNA Sample Prep Kit (Illumina-compatible), using 100 ng of fosmid DNA per well as the starting material. The 96 bar-coded Nextera libraries were pooled and sequenced on two lanes of an Illumina GAII (paired-end, 2 x 76-bp reads, with an additional 9-bp index read). Reads were mapped to the genome and analyzed as described in the supplementary information.

The authors report:

"We identified 4.1 million ‘singly unique nucleotide’ positions informative in distinguishing specific copies…these data identify human-specific expansions in genes associated with brain development, reveal extensive population genetic diversity, and detect signatures consistent with gene conversion in the human species. Our approach makes ~1000 genes accessible to genetic studies of disease association."

Sudmant, P. et al. (2010). Diversity of Human Copy Number Variation and Multicopy Genes Science, 330 (6004), 641-646 DOI: 10.1126/science.1197005