Publication
Detection and removal of barcode swapping in single-cell RNA-seq data
Publisher:
Cold Spring Harbor Laboratory
Date:
16-08-2017
DOI:
10.1101/177048
Abstract: Barcode swapping results in the mislabeling of sequencing reads between multiplexed s les on the new patterned flow cell Illumina sequencing machines. This may compromise the validity of numerous genomic assays, especially for single-cell studies where many s les are routinely multiplexed together. The severity and consequences of barcode swapping for single-cell transcriptomic studies remain poorly understood. We have used two statistical approaches to robustly quantify the fraction of swapped reads in each of two plate-based single-cell RNA sequencing datasets. We found that approximately 2.5% of reads were mislabeled between s les on the HiSeq 4000 machine, which is lower than previous reports. We observed no correlation between the swapped fraction of reads and the concentration of free barcode across plates. Furthermore, we have demonstrated that barcode swapping may generate complex but artefactual cell libraries in droplet-based single-cell RNA sequencing studies. To eliminate these artefacts, we have developed an algorithm to exclude in idual molecules that have swapped between s les in 10X Genomics experiments, exploiting the combinatorial complexity present in the data. This permits the continued use of cutting-edge sequencing machines for droplet-based experiments while avoiding the confounding effects of barcode swapping.