KAPA Adapters

• Index hopping
• Cross-contamination of barcodes or samples
• Template switching during PCR amplification of pooled samples
• Sequencing or analysis errors

Some of these issues can be mitigated through careful adapter design and quality control.

KAPA UDI Adapters are designed to support confident results through:

• Unique dual-index combinations that enable reads with unexpected barcode combinations to be filtered out prior to data analysis
• Stringent manufacturing procedures and sequencing-based QC testing to reduce the potential for index misassignment resulting from barcode cross-contamination

Figure 1. Unique dual indexing mitigates the impact of index misassignment during multiplexed sequencing on patterned flow cells.

Each dot in this dataset represents a PCR-free human whole-genome library, sequenced in pools of 8 (black) or 24 (blue) on Illumina HiSeq X instruments. Contamination rates, calculated with VerifyBamID, reflect potential sample cross-contamination based on genotype analysis. Index hopping contributes to observed contamination rates, and was significantly reduced after implementing the non-redundant dual indexing strategy and barcodes utilized in KAPA UDI Adapters (instead of the original single indexing workflow). Green lines represent the mean and red lines the upper and lower statistical control limits of the data, analyzed with JMP. Data courtesy of the Broad Institute (Cambridge, MA, USA).

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 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 incorrect 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.*

*Data courtesy of the Broad Institute (Cambridge, MA, USA).

KAPA Adapters deliver high and uniform conversion rates for libraries prepared from both Covaris-sheared and enzymatically fragmented DNA. This consistency demonstrates robust adapter performance across different library preparation workflows and supports reliable results for a wide range of sequencing applications.

Figure 3. Sequencing data were down-sampled to 27 million reads per technical replicate before sequence coverage calculation with Bedtools.

KAPA libraries prepared from Covaris-sheared and enzymatically fragmented DNA yielded a similar and fairly narrow distribution around an overall higher average coverage depth than Nextera libraries, which contained regions of low coverage and coverage hotspots.

Even limited library amplification can introduce bias, leading to uneven coverage and increased sequencing requirements—particularly for human and other complex genomes, or for microbial genomes with extreme GC content.

PCR-free workflows are now the gold standard for human whole-genome sequencing (WGS).

High-quality, full-length KAPA UDI Adapters, used together with KAPA EvoPrep Kits and KAPA EvoPlus V2 Kits, support the flexible and highly efficient library construction protocols needed for high-throughput, PCR-free human WGS.

KAPA Adapters are a collection of high-quality, UDI adapters for ligation-based library construction. They support a wide range of sequencing multiplexing levels (from 2-plex up to 384-plex) on both two- and four-channel Illumina instruments that use either patterned or non-patterned flow cells.

• Mitigate the impact of index misassignment with full-length KAPA UDI Adapters, offering 96 adapters with non-redundant index combinations, or with KAPA Universal Adapters plus KAPA UDI primer mixes
• Create up to 384 uniquely indexed libraries with truncated KAPA Universal Adapters (with or without UMIs) and KAPA UDI primer mixes