Preferential amplification of repetitive DNA during Next Generation Sequencing library creation of ancient DNA

Repetitive microsatellite DNA forms a universal component of eukaryote genomes and specific biochemical properties of such repeat regions may influence the outcome of laboratory protocols. The Atlantic cod (Gadus morhua) genome contains an order of magnitude more dinucleotide repeats than the majority of vertebrates, with over eight percent of its genome that can be classified as either AC or AG dinucleotide repeat. We find that the abundance of these repeats can be inflated in ancient DNA (aDNA) whole genome sequencing (WGS) data generated from this species, in particular in samples with a lower fragment length. This inflation is suppressed by a reduced number of amplification cycles and by the inclusion of manufactured dinucleotide repeat oligonucleotides during amplification. These data indicate that a biased amplification reaction leads to artificially high levels of AC and AG repeats. This process appears to be particularly efficient in Atlantic cod –likely due to its high genomic content of repeats with relatively simple sequence complexity. While the extend of such bias in other studies is unclear, we nonetheless urge caution when quantifying repeat content in aDNA WGS data, given that amplification bias can be difficult to detect if this process affects more complex repeat structures than dinucleotide repeats

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

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