By offering high sequencing speed and ultra-high-throughput at a low price, Illumina next-generation sequencing platforms have been widely adopted in recent years. However, an experiment with multiplexed library could be at risk of molecular recombination, known as “index switching”, which causes a proportion of the reads to be assigned to an incorrect sample. It is reported that a new advance, exclusion amplification (ExAmp) in conjunction with the patterned flow cell technology introduced on HiSeq 3000/HiSeq 4000/HiSeq X sequencing systems, potentially suffers from a higher rate of index switching than conventional bridge amplification.
University of Oslo researchers took advantage of the diverse but highly cell-specific expression of antigen receptors on immune cells to quantify index switching on single cell RNA-seq data that were sequenced on HiSeq 3000 and HiSeq 4000. By utilizing the unique antigen receptor expression, they could quantify the spread-of-signal from many different wells (n = 55 from total of three batches) due to index switching. Based on full-length T cell receptor (TCR) sequences from all samples reconstructed by TraCeR and TCR gene expression quantified by Kallisto, they found index switching in all three batches of experiments investigated. The median percentage of incorrectly detected markers was estimated to be 3.9% (interquartile range (IQR): 1.7%-7.3%). The researchers did not detect any consistent patterns of certain indices to be more prone to switching than others, suggesting that index switching is a stochastic process. These results confirm that index switching is a problem that affects samples run in multiplexed libraries on Illumina HiSeq 3000 and HiSeq 4000 platforms.
BCR reconstructed by BraCeR further verifies index switching
Expression of BraCeR-reconstructed BCR sequence “IGLV7-43_CTCAGGTCCCGTGGGT_IGLJ3” in Plate 4 and Plate 5. Two plates are bound by common column indices. Upper 8 rows labeled with only letters represent Plate 4; bottom 8 rows labeled with letter plus star are from Plate 5. The indices used are given in the brackets. Cell type is labeled in the corresponding well, T for T cell, Ps for source plasma cell, P for plasma cell, 0 for empty, 50 for mixture of multiple cells and X for unknown type.