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Length and GC-biases during sequencing library amplification: A comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries
Jesse Dabney and Matthias Meyer
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Supplementary Material

Figure 3. Average Duplicate Number versus Length in ancient DNA sequencing libraries. (Click to enlarge)

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.

Figure 4. Average Duplicate Number vs GC-content in ancient DNA sequencing libraries. (Click to enlarge)

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.

Competing interests

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]

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