Length and GC-biases during sequencing library amplification: a comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries

Biotechniques. 2012 Feb;52(2):87-94. doi: 10.2144/000113809.


High-throughput sequencing technologies frequently necessitate the use of PCR for sequencing library amplification. PCR is a sometimes enigmatic process and is known to introduce biases. Here we perform a simple amplification-sequencing assay using 10 commercially available polymerase-buffer systems to amplify libraries prepared from both modern and ancient DNA. We compare the performance of the polymerases with respect to a previously uncharacterized template length bias, as well as GC-content bias, and find that simply avoiding certain polymerase can dramatically decrease the occurrence of both. For amplification of ancient DNA, we found that some commonly used polymerases strongly bias against amplification of endogenous DNA in favor of GC-rich microbial contamination, in our case reducing the fraction of endogenous sequences to almost half.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Composition / genetics*
  • Buffers
  • DNA-Directed DNA Polymerase / genetics*
  • Gene Library*
  • Humans
  • Nucleic Acid Amplification Techniques / methods*
  • Sequence Analysis, DNA / methods*


  • Buffers
  • DNA-Directed DNA Polymerase