Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes – University of Copenhagen

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30 October 2013

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

New paper in PLoS ONE.

Second-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. However, these platforms are dependent on library construction and amplification steps that may result in sequences that do not reflect the original DNA template composition.

In this study, researchers from the Willerslev and Orlando groups, processing samples provided by colleagues in Denmark, France, Spain, Chile and Argentina, have shown that library preparation procedures based on AT-overhang adapter ligation introduce significant bias in the base composition of Illumina libraries.

This type of library building corresponds to the standard Illumina procedure. Therefore, the nature of the bias discovered in this study impacts ancient DNA but more generally, the field of genomics, due to the fact that the vast majority of genomes are sequenced nowadays on Illumina platforms.

This bias results in an uneven representation of different genomic landscapes, which has important consequences in structuring the lateral coverage of genome datasets sequenced at low depth, eg. in projects involving single cell genome sequencing, non-invasive samples, museomics and ancient DNA where DNA material is scarce.

This study also reveals that molecular tools currently available significantly limit our ability to successfully integrate into libraries the whole molecular complexity of ancient templates preserved in ancient DNA extracts. Additionally, the researchers demonstrate that library preparation procedures based on AT-overhang adapter ligation substantially change the shape of nucleotide misincorporation patterns.

Since the latter are used as important authentication criteria in ancient DNA research, these findings will help defining better guidelines for validating next-generation sequencing as truly authentic (and not contamination by-products).

The work has been published online in PLoS ONE 29. October 2013.

Authors

Seguin-Orlando A, Schubert M, Clary J, Stagegaard J, Alberdi MT, Prado JL, Prieto A, Willerslev E, Orlando L.