Estimating the rate of index hopping on the Illumina HiSeq X platform

AbstractThe high-throughput capacities of the Illumina sequencing platforms and 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. Alarmingly high rates of read misassignment of up to 10% were reported for the latest generation of lllumina sequencing machines. This potentially calls into question previously generated results and may make future use of the newest generation of platforms prohibitive. In this study we rely on barcodes, short sequences that are directly ligated to both ends of the DNA insert, which allows us to quantify the amount of index hopping. Correcting for multiple sources of noise, we identify on average only 0.470% of reads containing a hopped index. Multiplexing of samples on this platform is therefore unlikely to cause markedly different results to those obtained from older platforms.