Genome Biology and Evolution
Genome Biology and Evolution
5 years ago

Illumina library preparation for sequencing the GC-rich fraction of heterogeneous genomic DNA.

Fidel Botero-Castro, Benoit Nabholz, Marie-Ka Tilak, Nicolas Galtier
Standard Illumina libraries are biased towards sequences of intermediate GC-content. This results in an underrepresentation of GC-rich regions in sequencing projects of genomes with heterogeneous base composition, such as mammals and birds. We developed a simple, cost-effective protocol to enrich sheared genomic DNA in its GC-rich fraction by subtracting AT-rich DNA. This was achieved by heating DNA up to 90 °C before applying Illumina library preparation. We tested the new approach on chicken DNA and found that heated DNA increased average coverage in the GC-richest chromosomes by a factor up to six. Using a Taq polymerase supposedly appropriate for PCR amplification of GC-rich sequences had a much weaker effect. Our protocol should greatly facilitate sequencing and resequencing of the GC-richest regions of heterogeneous genomes, in combination with standard short-read and long-read technologies.

Publisher URL: http://doi.org/10.1093/gbe/evy022

DOI: 10.1093/gbe/evy022

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