Abstract
The high throughput capacities of the Illumina sequencing platforms and the possibility to label samples with unique identifiers has encouraged a wide use of sample multiplexing. However, this practice results in low rates of read misassignment (<1%) across samples sequenced on the same lane on all Illumina sequencing platforms that rely on the traditional bridge amplification. Alarmingly high rates of read misassignment of up to 10% were recently reported for the newest Illumina machines (HiSeq X and HiSeq 4000). This potentially calls into question previously generated and published results and may make future use of these platforms prohibitive for many applications in biology and medicine. In this study we rely on inline barcodes that are ligated to both ends of the DNA insert, to directly quantify the amount of index hopping in historical museum-preserved samples. As the barcodes become part of the sequencing read, they allow us to reliably infer the read origin even in the presence of index hopping. After sequencing the same pooled library of seven samples on three independent HiSeq X lanes and accounting for multiple possible sources of error, including barcode and index cross-contamination, we identified on average only 0.470% hopped reads. We conclude that index hopping happens on the newest generation of Illumina sequencing platforms, but results in a similar rate of read missagnment as reported for older Illumina machines. We nonetheless recommend using inline barcodes in multiplexing studies that rely on low-coverage data, require absolute certainty and/or aim to characterize rare variants.
