Effect 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.
Laboratory of Malaria and Vector Research, National Institutes of Health, Bethesda, MD, USA.
María José
MJ
López-Barragán
Mariam
M
Quiñones
Kairong
K
Cui
Jacob
J
Lemieux
Keji
K
Zhao
Xin-Zhuan
XZ
Su
D020856:AT Rich Sequence; D001482:Base Composition; D018503:Genome, Protozoan / Q000235:genetics; D055106:Genome-Wide Association Study / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D010963:Plasmodium falciparum / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D013696:Temperature