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Effect of PCR extension temperature on high-throughput sequencing.
Mol Biochem Parasitol. 2011 Mar;176(1):64-7
Authors: López-Barragán MJ, Quiñones M, Cui K, Lemieux J, Zhao K, Su XZ
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.
PMID: 21112355 [PubMed - indexed for MEDLINE]
More...
Effect of PCR extension temperature on high-throughput sequencing.
Mol Biochem Parasitol. 2011 Mar;176(1):64-7
Authors: López-Barragán MJ, Quiñones M, Cui K, Lemieux J, Zhao K, Su XZ
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.
PMID: 21112355 [PubMed - indexed for MEDLINE]
More...