title: Palindromic sequence artifacts generated during next generation sequencing library preparation from historic and ancient DNA.
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.
authors.affiliation: Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Bastiaan
authors.initials: B
authors.lastname: Star
authors.affiliation: Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Nederbragt
authors.affiliation: Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Marianne H S
authors.initials: MH
authors.lastname: Hansen
authors.affiliation: Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Morten
authors.initials: M
authors.lastname: Skage
authors.affiliation: Department of Medical Genetics, Oslo University Hospital, Oslo, Norway.
authors.forename: Gregor D
authors.initials: GD
authors.lastname: Gilfillan
authors.affiliation: Fisheries and Oceans Canada, St. John's, Newfoundland, Canada.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Bradbury
authors.affiliation: Marine Research Institute, Reykjavík, Iceland.
authors.forename: Christophe
authors.initials: C
authors.lastname: Pampoulie
authors.affiliation: Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Nils Chr
authors.initials: NC
authors.lastname: Stenseth
authors.affiliation: Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Kjetill S
authors.initials: KS
authors.lastname: Jakobsen
authors.affiliation: Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Sissel
authors.initials: S
authors.lastname: Jentoft
mesh_terms: D000818:Animals; D016477:Artifacts; D004247:DNA / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D004277:DNA, Single-Stranded; D004247:DNA