The efficacy of high-throughput sequencing and target enrichment on charred archaeobotanical remains

The majority of archaeological plant material is preserved in a charred state and obtaining reliable ancient DNA data from these remains has presented challenges due to problems associated with high rates of nucleotide damage, short DNA fragment lengths, low endogenous DNA content and the potential for modern contamination. It has been suggested that high-throughput sequencing (HTS) technologies coupled with DNA capture enrichment techniques have the potential to overcome some of these limitations. Here we report the findings of HTS and target enrichment on four important archaeological crops (barley, grape, maize and rice) performed in three different laboratories, presenting the largest HTS assessment of charred archaeobotanical specimens to date. Rigorous analysis of our data indicated a lack of endogenous DNA in nearly all samples, with the exception of one lightly-charred maize cob. Even with target enrichment, this sample failed to yield adequate data required to address fundamental questions in archaeology and biology. We further reanalysed part of an existing dataset on charred plant material, and found all purported endogenous DNA sequences were likely to be spurious. We suggest these technologies are not suitable for use with charred archaeobotanicals and urge great caution when interpreting HTS data generated from these remains.