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  • #1

    Ligation Biases during library construction

    Has anyone else experienced this base composition bias during ligation? Check out figures 1 & 2:


    Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

    Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes
    http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0078575
    Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.


    - Genohub
    Tags: adapter composition bias, ligation bias
  • #2
    I have not looked at this but they use a library prep method where the dNTPs are not removed before A-tailing. This reminds me of a poster from Life where dNTPs where shown to cause similar bias against inserts starting with T/C:

    Thermo Fisher Scientific - US
    http://www3.appliedbiosystems.com/cms/groups/global_marketing_group/documents/generaldocuments/cms_091610.pdf
    Thermo Fisher Scientific enables our customers to make the world healthier, cleaner and safer. Delivering technology, pharmaceutical and biotechnology services.

    Comment

    • #3
      Interesting poster. This paper also compares blunt ended vs. AT ligation. It would nice to see whether any of the combined End Repair / Adenylation steps that use just a polymerase to A-tail (and have to deal with a full assortment of dNTPS, rather than just dA) are also introducing bias or just reducing preparation efficiency.

      - Genohub

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