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. Obtaining reliable ancient DNA data from these remains has presented challenges due to 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 enrichment techniques may 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-excluding false-positives due to background contamination or incorrect index assignments-indicated a lack of endogenous DNA in nearly all samples, except for 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 data obtained by HTS of these remains.