Palindromic sequence artifacts generated during next generation sequencing library preparation from historic and ancient DNA

PLoS One. 2014 Mar 7;9(3):e89676. doi: 10.1371/journal.pone.0089676. eCollection 2014.

Abstract

Degradation-specific processes and variation in laboratory protocols can bias the DNA sequence composition from samples of ancient or historic origin. Here, we identify a novel artifact in sequences from historic samples of Atlantic cod (Gadus morhua), which forms interrupted palindromes consisting of reverse complementary sequence at the 5' and 3'-ends of sequencing reads. The palindromic sequences themselves have specific properties - the bases at the 5'-end align well to the reference genome, whereas extensive misalignments exists among the bases at the terminal 3'-end. The terminal 3' bases are artificial extensions likely caused by the occurrence of hairpin loops in single stranded DNA (ssDNA), which can be ligated and amplified in particular library creation protocols. We propose that such hairpin loops allow the inclusion of erroneous nucleotides, specifically at the 3'-end of DNA strands, with the 5'-end of the same strand providing the template. We also find these palindromes in previously published ancient DNA (aDNA) datasets, albeit at varying and substantially lower frequencies. This artifact can negatively affect the yield of endogenous DNA in these types of samples and introduces sequence bias.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Artifacts*
  • DNA / genetics*
  • DNA, Single-Stranded / genetics
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Sequence Analysis, DNA / methods

Substances

  • DNA, Single-Stranded
  • DNA

Grant support

This work was supported by the Norwegian Research Council under the project, “Fisheries induced evolution in Atlantic cod investigated by ancient and historic samples (#203850/E40)”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.