Question:Is there evidence of index hopping occurring in 10x Genome libraries?
Answer: Index hopping or index switching is a known phenomenon that has impacted NGS technologies from the time sample multiplexing was developed. It causes a specific type of misassignment that results in the incorrect assignment of libraries from the expected index to a different index (in the multiplexed pool)(definition from Illumina website).
Index hopping may occur when adapters and primers are not efficiently removed by the SPRI bead clean-up steps during the workflow. We have not observed evidence of index hopping when sequencing 10x genome libraries. This doesn't preclude the possibility that it occurs but the design of the 10x genome assay scheme is such that the probability of switching events is extremely low.
In general, the Chromium Genome and Exome libraries are free of these leftover adapters and primers, significantly reducing the chances of index hopping to occur on the Illumina flowcell. Moreover, because evidence from multiple overlapping sequence reads is required to call any event (SNP, gene phasing, gene fusion, etc.) more than one index hopping event would have to occur for library molecules from the same genetic locus to cause a significant effect in the data.
A typical Genome sample utilizes ~1 million 10x barcodes out of 4.4 million total. The chance of a molecule switching to a new sample index and having a 10x barcode from the destination sample is reduced by a factor of 4. In addition, downstream Long Ranger analysis uses both the 10x barcode and the genome alignment to determine which GEM or partition the molecule came from, further reducing the likelihood of index switching from affecting the analysis. Assuming a 50 kb fragment length and 3.2 Gb genome size, the likelihood of two molecules sharing the same genomic locus in a single GEM are 1/7000.