Short technical reportEffect of PCR extension temperature on high-throughput sequencing

The DNA amplification process can be a source of bias and artifacts, especially when amplifying genomic areas with extreme AT or GC content. The human malaria parasite Plasmodium falciparum has an AT-rich genome, and some of its highly AT-rich regions have been shown to be refractory to polymerase chain reaction (PCR) amplification. Biased amplification may lead to erroneous conclusions for studies investigating genome-wide gene expression, nucleosome position, and copy number variation. Here we compare genome-wide nucleosome coverage in libraries amplified at three different extension temperatures and show that reduction in PCR extension temperature from 70 °C to 60 °C can greatly increase the fraction of coverage at AT-rich regions of the P. falciparum genome. Our method will improve the efficiency and coverage in sequencing an AT-rich genome.

Graphical abstract. Reduced PCR extension temperature increases high throughput sequencing coverage at AT-rich regions.Download high-res image (82KB)Download full-size imageResearch highlights▶ Sequence coverages amplified at extension temperatures of 70 °C, 65 °C, and 60 °C were compared. ▶ Significantly increased sequence coverage at AT-rich regions when amplified at 60 °C. ▶ DNA with a wide range of AT content can be amplified reliably using an extension temperature of 60 °C.