Abstract

Ancient DNA research has developed rapidly over the past few decades due to the improvement in PCR and next-generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from the raw sequencing data. This is often difficult due to the degradation of ancient DNA and high levels of contamination, especially homologous contamination. In this study, we collected whole genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from the homologous contaminations, we attempted to recover reads based on the ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing the homologous contaminations to a very low proportion. Overall, these recommendations for ancient DNA mapping and separation of endogenous DNA in this study could facilitate future studies of ancient DNA.

Highlights

  • Ancient DNA research provides direct evidence to reconstruct prehistoric biogeographies and biodiversities, which can further help to explain long-standing questions in evolution, phylogeny, taxonomy, and adaptations (Chang et al, 2017; Delsuc et al, 2019; Palkopoulou et al, 2018; Sikora et al, 2019; Stoneking & Krause, 2011)

  • The comparison was achieved by calculating the contamination rate after treatment (CRT), the loss rate of endogenous DNA (LRE), and the running time of mapping (MT) for each dataset, and performing Repeated Measurement Analysis of Variance

  • We further evaluated the influence of different samples and different contamination rates on ancient DNA mapping

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Summary

| INTRODUCTION

Ancient DNA research provides direct evidence to reconstruct prehistoric biogeographies and biodiversities, which can further help to explain long-standing questions in evolution, phylogeny, taxonomy, and adaptations (Chang et al, 2017; Delsuc et al, 2019; Palkopoulou et al, 2018; Sikora et al, 2019; Stoneking & Krause, 2011). In the mapping procedure for ancient DNA, the software BWA (Li & Durbin, 2009) with parameters set “aln -l 1,024 -n 0.03” is usually applied to map ancient sequencing data against the reference genome (Schubert et al, 2012). Skoglund et al (2014) developed PMDtools to separate genuine endogenous DNA from homologous contaminations This method is effective in filtering modern human contaminated DNA from ancient human DNA. We collected whole-genome sequencing data generated by the Illumina Hiseq platform from 6 samples (representing three species) to optimize ancient DNA mapping. This step is critical to improving the mapping rate of endogenous ancient DNA. We sought to develop mapping guidelines that, when coupled with screening recommendations to control for modern DNA contamination, could increase the effectiveness of future studies of ancient DNA

| MATERIALS AND METHODS
| DISCUSSION
Findings
| CONCLUSION
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