Abstract
Ancient DNA research has been revolutionized following development of next-generation sequencing platforms. Although a number of such platforms have been applied to ancient DNA samples, the Illumina series are the dominant choice today, mainly because of high production capacities and short read production. Recently a potentially attractive alternative platform for palaeogenomic data generation has been developed, the BGISEQ-500, whose sequence output are comparable with the Illumina series. In this study, we modified the standard BGISEQ-500 library preparation specifically for use on degraded DNA, then directly compared the sequencing performance and data quality of the BGISEQ-500 to the Illumina HiSeq2500 platform on DNA extracted from 8 historic and ancient dog and wolf samples. The data generated were largely comparable between sequencing platforms, with no statistically significant difference observed for parameters including level (P = 0.371) and average sequence length (P = 0718) of endogenous nuclear DNA, sequence GC content (P = 0.311), double-stranded DNA damage rate (v. 0.309), and sequence clonality (P = 0.093). Small significant differences were found in single-strand DNA damage rate (δS; slightly lower for the BGISEQ-500, P = 0.011) and the background rate of difference from the reference genome (θ; slightly higher for BGISEQ-500, P = 0.012). This may result from the differences in amplification cycles used to polymerase chain reaction–amplify the libraries. A significant difference was also observed in the mitochondrial DNA percentages recovered (P = 0.018), although we believe this is likely a stochastic effect relating to the extremely low levels of mitochondria that were sequenced from 3 of the samples with overall very low levels of endogenous DNA. Although we acknowledge that our analyses were limited to animal material, our observations suggest that the BGISEQ-500 holds the potential to represent a valid and potentially valuable alternative platform for palaeogenomic data generation that is worthy of future exploration by those interested in the sequencing and analysis of degraded DNA.
Highlights
As with many other disciplines, the advent of next-generation sequencing (NGS) platforms has revolutionized ancient DNA research
The data generated were largely comparable between sequencing platforms, with no statistically significant difference observed for parameters including level (P = 0.371) and average sequence length (P = 0718) of endogenous nuclear DNA, sequence GC content (P = 0.311), double-stranded DNA damage rate (v. 0.309), and sequence clonality (P = 0.093)
We acknowledge that our analyses were limited to animal material, our observations suggest that the BGISEQ-500 holds the potential to represent a valid and potentially valuable alternative platform for palaeogenomic data generation that is worthy of future exploration by those interested in the sequencing and analysis of degraded DNA
Summary
As with many other disciplines, the advent of next-generation sequencing (NGS) platforms has revolutionized ancient DNA (aDNA) research. At least the initial stages of the library construction method underlying the BGISEQ-500 are sufficiently close to the methods currently used for Illumina palaeogenomic sequencing, and can be modified based upon some of the abovementioned previous aDNA-related developments if needed To fully explore this platform’s potential for aDNA, we undertook a direct performance comparison against Illumina technology by building libraries and sequencing 8 historic and ancient DNA extracts. To both keep the underlying variables as similar as possible and to exploit a recent (Illumina-based) methodological development that (i) simplifies library construction and minimizes hands-on time and economic cost [13] and (ii) performs at least as well as the Meyer and Kircher [12] blunt end ligation method that many palaeogenomicists favour, we did not use the original BGISEQ500 library method, but rather developed a new protocol based on our recently developed blunt end single tube (BEST) method [13]. Following DNA extraction, 2 aliquots of each extract were constructed in the Illumina and BGISEQ-500 libraries, respectively, using identical amounts of starting material (16.3 μl, ∼5– 50 ng DNA input sample dependent), and subsequently sequenced to enable bioinformatic comparisons on the data
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