9/12/2023 0 Comments Complementary sequence calculator![]() For instance, the GC content is increased for fragments of shorter length –, varying polymerases affect template length and GC content, bias against fragments starting with thymines residues has been observed using AT-overhang ligation protocols and the typical complementary G-to-A substitutions at 3′-ends are absent when using protocols targeting single-stranded DNA (ssDNA). Additionally, varying molecular approaches in next generation library creation protocols can further influence base composition of aDNA. Specifically, enhanced cytosine deamination in single stranded 5′-overhangs leads to C-to-T substitutions at 5′-ends of sequences, and complementary G-to-A substitutions at 3′-ends –, whereas fragmentation bias is evident through a relative increase of purines at the positions immediately preceding the 5′ termini. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.Įxtensive degradation processes alter the base composition of historic and ancient DNA sequence data through biased sequence substitution and fragmentation. ![]() This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: 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)”. Received: NovemAccepted: JanuPublished: March 7, 2014Ĭopyright: © 2014 Star et al. PLoS ONE 9(3):Įditor: Ludovic Orlando, Natural History Museum of Denmark, University of Copenhagen, Denmark (2014) Palindromic Sequence Artifacts Generated during Next Generation Sequencing Library Preparation from Historic and Ancient DNA. This artifact can negatively affect the yield of endogenous DNA in these types of samples and introduces sequence bias.Ĭitation: Star B, Nederbragt AJ, Hansen MHS, Skage M, Gilfillan GD, Bradbury IR, et al. We also find these palindromes in previously published ancient DNA (aDNA) datasets, albeit at varying and substantially lower frequencies. 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. 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. 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. 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. ![]() Degradation-specific processes and variation in laboratory protocols can bias the DNA sequence composition from samples of ancient or historic origin.
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