Identification and quantification of chimeric sequencing reads in a highly multiplexed RAD‐seq protocol

Highly multiplexed approaches have become common in genomic studies. They have improved the cost‐effectiveness of genotyping hundreds of individuals using combinatorially barcoded adapters. These strategies, however, can potentially misassigned reads to incorrect samples. Here, we used a modified quaddRAD protocol to analyse the occurrence of index hopping and PCR chimeras in a series of experiments with up to 100 multiplexed samples per sequencing lane (639 samples in total). We created two types of sequencing libraries: four libraries of type A, where PCRs were run on individual samples before multiplexing, and three libraries of type B, where PCRs were run on pooled samples. We used fixed pairs of inner barcodes to identify chimeric reads. Type B libraries show a higher percentage of misassigned reads (1.15%) than type A libraries (0.65%). We also quantify the commonly undetectable chimeric sequences that occur whenever multiplexed groups of samples with different outer barcodes are sequenced together on a single flow cell. Our results suggest that these types of chimeric sequences represent up to 1.56% and 1.29% of reads in type A and B libraries, respectively. We also show that increasing the number of mismatches allowed for barcode rescue to above 2 dramatically increases the number of recovered chimeric reads. We provide recommendations for developing highly multiplexed RAD‐seq protocols and analysing the resulting data to minimize the generation of chimeric sequences, allowing their quantification and a finer control on the number of PCR cycles necessary to generate enough input DNA for library preparation.