Figure 2. Sequencing-based QC testing of KAPA Adapters for barcode cross-contamination improves confidence in results.
In this in-house developed QC assay, each KAPA UDI Adapter is ligated to a unique, synthetic linear insert. The 96 libraries are pooled and sequenced on an Illumina NextSeq™ 500 instrument. Data are subsampled to 500,000 reads per library, before adapter trimming and alignment to the synthetic reference sequences. Aligned bam files are downsampled to the lowest aligned read count for final calculations. The heat map shown here is a representative barcode cross-contamination test result for KAPA UDI Adapters, and shows the percentage of reads associated with each insert (columns) and barcode (rows) combination. Dark green blocks across the diagonal correspond to correct UDI-insert combinations. Every other block corresponds to the percentage of reads for a particular insert associated with one of the other expected index combinations in the set of 96, and is colored according to the scale given on the right. The test confirms that adapters are plated in the correct wells, and that index misassignment attributable to cross-contamination with UDI combinations that can’t be filtered out is extremely low compared to misassignment from other potential sources, such as index hopping.1 A similar in-house developed QC assay is followed for the KAPA UDI Primer Mixes.