Through extensive simulations we show that even low index hopping rates can lead to biases in ancient DNA studies when multiplexing samples with vastly different quantities of endogenous material.

The high-throughput capacities of the Illumina sequencing platforms and the possibility to label samples individually have encouraged a wide use of sample multiplexing. However, this practice results in read misassignment (usually <1%) across samples sequenced on the same lane. The specific quantity of misassigned reads depends on the number of reads that any given sample contributes to the total sequencing pool, so that samples with few sequenced reads receive the greatest proportion of misassigned reads. This particularly affects ancient DNA samples, as these frequently differ in endogenous content. We simulated ancient DNA sequencing reads under varying rates of index-hopping (described in van der Valk et. al (2019) Index hopping on the Illumina HiseqX platform and its consequences for ancient DNA studies) to asses biases introduced as a consequence of index-hopping. The library names are as follows: X%-samplename-coverage-endogenous-content.fastq.gz Were X is the simulated rate of index-hopping, samplename the species and individual name, coverage is depicted ending with X and endogenous content represents the fraction of endogenous reads in the simulated library.