While all polymerases exhibited inefficiencies in amplifying library molecules with low (<30%) or high (>70%) GC-content, biases were smallest with AccuPrime Pfx (Figure 4). Phusion HF, and to a lesser extent AmpliTaq Gold, showed a very pronounced bias toward molecules with >50% GC, which is in line with the results obtained from modern human DNA above. Since microbial DNA in this ancient sample is characterized by high GC-content (∼64%), this explains the higher fraction of microbial sequences observed in these libraries. Generally, looking at average duplicate numbers provides a higher resolution of the GC bias than the analysis performed with modern DNA.
In this study we identified PCR polymerases as a main source of both length and GC-content bias in modern human and short-insert ancient sequencing libraries. Under the parameters tested, Phusion polymerases in HF buffer and AmpliTaq Gold consistently introduced dramatic biases in both types of libraries, while the biases introduced by the other four polymerases are more subtle. For short-insert ancient DNA libraries, AccuPrime Pfx leads to a higher percentage of endogenous sequences while maintaining the length and GC-content profile of the input library. Furthermore, we found no dramatic effect on either length or GC-content bias when amplifying into PCR plateau.
We should note that this was a naïve approach using only the manufacturers’ polymerase-buffer systems and suggested parameters. Other factors, such as PCR additives and optimized thermocycling parameters, have been shown to boost performance when dealing with GC-biases (11). In this direction, limiting dilutions and counting PCR duplicates provides a very simple assay tool for detecting PCR biases, and can be used by any lab to characterize and optimize their preferred polymerase.
We would like to thank Susanna Sawyer for providing the Neandertal library, Martin Kircher for data processing, the Sequencing Group for help with sequencing and, finally, Svante Pääbo and the Dept. of Evolutionary Genetics for helpful discussion. This work was supported by the Max Planck Society.
The authors declare no competing interests.
Address correspondence to Jesse Dabney, Max Planck Institute for Evolutionary Anthropology, Department of Evolutionary Genetics, 04103 Leipzig, Germany. Email: [email protected]
1.) McPherson, M.J., and S.G. Moller. 2006. PCR. Taylor & Francis Group, New York, NY.[CrossRef] [PubMed] 2.) Kanagawa, T. 2003. Bias and artifacts in multitemplate polymerase chain reactions (PCR). J. Biosci. Bioeng. 96:317-323.[CrossRef] [PubMed] 3.) Day, D.J., P.W. Speiser, E. Schulze, M. Bettendorf, J. Fitness, F. Barany, and P.C. White. 1996. Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees. Hum. Mol. Genet. 5:2039-2048.[CrossRef] [PubMed] 4.) Ogino, S., and R.B. Wilson. 2002. Quantification of PCR bias caused by a single nucleotide polymorphism in SMN gene dosage analysis. J. Mol. Diagn. 4:185-190.[CrossRef] [PubMed] 5.) Barnard, R., V. Futo, N. Pecheniuk, M. Slattery, and T. Walsh. 1998. PCR bias toward the wild-type k-ras and p53 sequences: implications for PCR detection of mutations and cancer diagnosis. Biotechniques 25:684-691.[CrossRef] [PubMed] 6.) Kozarewa, I., Z. Ning, M.A. Quail, M.J. Sanders, M. Berriman, D.J. Turner, and N. America. 2009. Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G + C) -biased genomes. Nat. Methods 6:291-295.[CrossRef] [PubMed] 7.) Margulies, M., M. Egholm, W.E. Altman, S. Attiya, J.S. Bader, L.A. Bemben, J. Berka. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376-380.[CrossRef] [PubMed] 8.) Hodges, E., M. Rooks, Z. Xuan, A. Bhattacharjee, D. Benjamin Gordon, L. Brizuela, W. Richard McCombie. 2009. Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nat. Protocols 4:960-974.[CrossRef] [PubMed] 9.) Mamanova, L., A.J. Coffey, C.E. Scott, I. Kozarewa, E.H. Turner, A. Kumar, E. Howard. 2010. Target-enrichment strategies for next-generation sequencing. Nat. Methods 7:111-118.[CrossRef] [PubMed] 10.) Sam, L.T., D. Lipson, T. Raz, X. Cao, J. Thompson, P.M. Milos, D. Robinson. 2011. A comparison of single molecule and amplification based sequencing of cancer transcriptomes. PLoS One 6:e17305.[CrossRef] [PubMed] 11.) Aird, D., M.G. Ross, W.S. Chen, M. Danielsson, T. Fennell, C. Russ, D.B. Jaffe. 2011. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol. 12:R18.[CrossRef] [PubMed] 12.) Pääbo, S. 1989. Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. Proc. Natl. Acad. Sci. USA 86:1939-1943.[CrossRef] [PubMed] 13.) Orlando, L., A. Ginolhac, and M. Raghavan. 2011. True single-molecule DNA sequencing of a pleistocene horse bone. Genome Res. 21:1705-1719.[CrossRef] [PubMed] 14.) Briggs, A.W., U. Stenzel, P.L.F. Johnson, R.E. Green, J. Kelso, K. Prüfer, M. Meyer. 2007. Patterns of damage in genomic DNA sequences from a Neandertal. Proc. Natl. Acad. Sci. USA 104:14616-14621.[CrossRef] [PubMed] 15.) Gibbons, A. 2011. Who were the Denisovans?. Science 333:1084-1087.[CrossRef] [PubMed] 16.) Rohland, N., and M. Hofreiter. 2007. Ancient DNA extraction from bones and teeth. Nat. Protocols 2:1756-1762.[CrossRef] [PubMed] 17.) Meyer, M., and M. Kircher. 2010. Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing. Cold Spring Harb Protoc..[CrossRef] [PubMed] 18.) Briggs, A.W., U. Stenzel, M. Meyer, J. Krause, M. Kircher, and S. Pääbo. 2010. Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA. Nucleic Acids Res. 38:e87.[CrossRef] [PubMed] 19.) Krause, J., Q. Fu, J.M. Good, B. Viola, M.V. Shunkov, A.P. Derevianko, and S. Pääbo. 2010. The complete mitochondrial DNA genome of an unknown hominin from southern Siberia. Nature 464:894-897.[CrossRef] [PubMed] 20.) Meyer, M., A.W. Briggs, T. Maricic, B. Höber, B. Höffner, J. Krause, A. Weihmann. 2008. From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing. Nucleic Acids Res. 36:e5.[CrossRef] [PubMed] 21.) Kircher, M., S. Sawyer, and M. Meyer. 2011. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Res. 40:1-8.[CrossRef] [PubMed] 22.) Kircher, M., U. Stenzel, and J. Kelso. 2009. Improved base calling for the Illumina Genome Analyzer using machine learning strategies. Genome Biol. 10:R83.[CrossRef] [PubMed] 23.) Li, H., and R. Durbin. 2010. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26:589-595.[CrossRef] [PubMed] 24.) Kircher, M., P. Heyn, and J. Kelso. 2011. Addressing challenges in the production and analysis of Illumina sequencing data. BMC Genomics 12:382.[CrossRef] [PubMed] 25.) Reich, D., R.E. Green, M. Kircher, J. Krause, N. Patterson, E.Y. Durand, B. Viola. 2010. Genetic history of an archaic hominin group from Denisova Cave in Siberia. Nature 468:1053-1060.[CrossRef] [PubMed] 26.) Rasmussen, M., Y. Li, S. Lindgreen, J.S. Pedersen, A. Albrechtsen, I. Moltke, M. Metspalu. 2010. Ancient human genome sequence of an extinct Palaeo-Eskimo. Nature 463:757-762.[CrossRef] [PubMed] 27.) Heyn, P., U. Stenzel, A.W. Briggs, M. Kircher, M. Hofreiter, and M. Meyer. 2010. Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA. Nucleic Acids Res. 38:e161.[CrossRef] [PubMed]