Double indexing improves the accuracy of multiplex sequencing on the Illumina platform by reducing sample misidentification. The study found that the rate of sample misidentification was as high as 0.3%, which is much higher than expected. This is primarily due to mixed clusters on the flow cell, where sequences from different templates are combined. Other factors, such as cross-contamination of indexed oligonucleotides and PCR jumping, also contribute to false index pairs. Double indexing, which uses indexes in both library adapters, helps to identify and eliminate these errors. The study also showed that using a quality filter on the index reads can further reduce false assignments. The results indicate that mixed clusters are the main source of false index pairs, and that double indexing significantly improves the accuracy of multiplex sequencing. The study also highlights the importance of quality filtering and the potential for cross-contamination in sequencing experiments. Overall, double indexing is a powerful method for improving the accuracy of multiplex sequencing on the Illumina platform.Double indexing improves the accuracy of multiplex sequencing on the Illumina platform by reducing sample misidentification. The study found that the rate of sample misidentification was as high as 0.3%, which is much higher than expected. This is primarily due to mixed clusters on the flow cell, where sequences from different templates are combined. Other factors, such as cross-contamination of indexed oligonucleotides and PCR jumping, also contribute to false index pairs. Double indexing, which uses indexes in both library adapters, helps to identify and eliminate these errors. The study also showed that using a quality filter on the index reads can further reduce false assignments. The results indicate that mixed clusters are the main source of false index pairs, and that double indexing significantly improves the accuracy of multiplex sequencing. The study also highlights the importance of quality filtering and the potential for cross-contamination in sequencing experiments. Overall, double indexing is a powerful method for improving the accuracy of multiplex sequencing on the Illumina platform.