Library Construction for Ancient Genomics: Single Strand or Double Strand?

Abstract

A novel method of library construction that takes advantage of a single-stranded DNA ligase has been recently described and used to generate high-resolution genomes from ancient DNA samples. While this method is effective and appears to recover a greater fraction of endogenous ancient material, there has been no direct comparison of results from different library construction methods on a diversity of ancient DNA samples. In addition, the single-stranded method is limited by high cost and lengthy preparation time and is restricted to the Illumina sequencing platform. Here we present in-depth comparisons of the different available library construction methods for DNA purified from 16 ancient and modern faunal and human remains, covering a range of different taphonomic and climatic conditions. We further present a DNA purification method for ancient samples that permits the concentration of a large volume of dissolved extract with minimal manipulation and methodological improvements to the single-stranded method to render it more economical and versatile, in particular to expand its use to both the Illumina and the Ion Torrent sequencing platforms. We show that the single-stranded library construction method improves the relative recovery of endogenous to exogenous DNA for most, but not all, of our ancient extracts.

Extending the spectrum of DNA sequences retrieved from ancient bones and teeth

“…In agreement with previous observations (Bennett et al 2014;Wales et al 2015;Gansauge et al 2017), the informative sequence content is substantially lower in the double-stranded than the single-stranded libraries (Supplemental Table S4; Supplemental Fig. S13).…”

Relative performance of two DNA extraction and library preparation methods on archaeological human teeth samples

“…Previous studies have provided evidence suggesting that the ssDNA method improves endogenous DNA yields and library complexity (Meyer et al 2012;Prüfer et al 2014;Bennett et al 2014). This is likely due to its ability to (i) help recover DNA molecules with single-stranded breaks and those with end modifications that are entirely lost during dsDNA construction, and (ii) because it avoids DNA loss during purification steps that are an integral part of the dsDNA construction protocol (Bennett et al 2014).…”

“…Previous studies have provided evidence suggesting that the ssDNA method improves endogenous DNA yields and library complexity (Meyer et al 2012;Prüfer et al 2014;Bennett et al 2014). This is likely due to its ability to (i) help recover DNA molecules with single-stranded breaks and those with end modifications that are entirely lost during dsDNA construction, and (ii) because it avoids DNA loss during purification steps that are an integral part of the dsDNA construction protocol (Bennett et al 2014). Although differences were observed in the percentage of endogenous DNA sequences retrieved for the different library preparation methods (Table 1), at the relatively low sequencing depth of our experiment these did not consistently favour one approach over the other, and differences were not found to be significant (paired t-test = -1.4773, df = 4, p-value = 0.8932).…”

“…In both cases, ideal methods should maximize the potential for sequencing endogenous DNA, at the exclusion of contaminants and exogenous sources of DNA. Although several promising protocols have been developed to address these goals (Yang et al 1998;Rohland and Hofreiter 2007;Meyer et al 2012;Dabney et al 2013), validation of their efficacy has been principally limited to a small number of samples with DNA of either very low (<1%) or very high (>30%) endogenous content (Meyer et al 2012;Bennett et al 2014;Gamba et al 2016;Barlow et al 2016). Therefore, as argued by Bennett et al (2014) and Barlow et al (2016), there is a need to expand the comparative performance of different methods across a wider range of endogenous DNA preservation and investigate the different effects that methods have on data generation.…”

Tracing the origin of our species through palaeogenomics

“…These immobilized molecules are then converted into double-stranded molecules, a new adaptor is added to the other end, and the molecules are amplified with a DNA polymerase using primers that correspond to the ligated adapters. This method increases significantly the yield of ancient DNA amenable to sequencing in the majority of ancient samples [12]. It was used to sequence the whole genome of the Denisovan from 10 mg of the pinky bone and to cover 99.9% of all nucleotide positions at least once, and 99.4% at least six times [2].…”