KAPA UDI Adapters

Overview

Full-length adapters are compatible with ligation-based library construction workflows for direct and targeted multiplexed DNA and RNA (cDNA) sequencing applications on the Illumina^® platform. Since sequencing barcodes are added during an adapter ligation step, full-length adapters are particularly well suited for PCR-free workflows.

KAPA Unique Dual-Indexed (UDI) Adapters have a standard Illumina adapter backbone. They are therefore easily incorporated in new and existing Illumina sequencing workflows, and do not require customized sequencing or indexing primers. A combination of 8-nt sequencing barcodes on each of the i5 and i7 adapter oligos supports paired-end sequencing on 1- , 2- and 4-channel Illumina instruments. KAPA UDI Adapters support a broad range of sample pooling (2- plex up to 96-plex, as appropriate for your workflow), for target enrichment and/or sequencing.

Unique dual-indexing is recommended for all applications and Illumina sequencers.^1,2

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Fewer misassigned reads improve confidence in results

Index misassignment during multiplexed sequencing may be the result of index hopping, barcode or sample cross-contamination, template switching during PCR amplification of pooled samples, and/or sequencing/analysis errors; some of which may be mitigated by adapter design and quality
Unique dual-index combinations in KAPA UDI Adapters allow reads with unexpected barcode combinations to be filtered out prior to data analysis
KAPA UDI Adapters are manufactured using stringent procedures and undergo sequencing-based QC testing to reduce the potential for index misassignment resulting from barcode cross-contamination

View data demonstrating how the unique dual index combinations used in KAPA UDI Adapters, as well as sequencing-based QC testing for barcode cross-contamination mitigate the potential impact of index misassignment.

Figure 1. Unique dual indexing mitigate 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. Everyother 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.

High-performance, complete workflows for a wide range of applications

High-performance library construction workflows require appropriate, high-quality accessories such as adapters and beads to achieve optimal library yields and quality.
KAPA UDI Adapters undergo functional testing in an NGS library construction workflow to confirm high levels of library construction efficiency, minimal levels of adapter-dimer formation and consistent performance across all 96 index combinations.

View data demonstrating that KAPA Adapters support efficient and consistent library construction efficiency across a range of different sample types, inputs and 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.

Flexible and efficient PCR-free human whole-genome sequencing

Even a small amount of library amplification may introduce bias which results in uneven coverage and the requirement for more sequencing; particularly when sequencing human and other complex genomes, or microbial genomes with extreme GC-content
PCR-free workflows have become the gold standard for human whole-genome sequencing (WGS)
High-quality, full-length KAPA UDI Adapters used in combination with KAPA HyperPrep Kits support the flexible and highly efficient library construction protocols needed for high-throughput, PCR-free human WGS

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References

https://www.illumina.com/science/education/minimizing-index-hopping.html. Accessed February 2019.
Costello M, et al. BMC Genomics. 2018;19:332

KAPA Unique Dual-Index (UDI) Adapters contain full-length, ready-to-use, QC-tested, dual-indexed Illumina adapters for ligation-based library construction. Kit specifications are listed below.

For additional details related to product shipping, storage and handling, please refer to technical documentation.

¹The sets of 192 and 768 barcode sequences used in KAPA UDI Adapters and KAPA UDI Primer Mixes (respectively) are exclusive to Roche and different between the two sets.

²Theoretically possible but not fully tested.

³This is sufficient for four or one library preps (KAPA UDI Adapter plate or KAPA UDI Primer Mixes plate, respectively) with the KAPA HyperPrep or KAPA HyperPlus Kit if no adapter dilution is required. Generous overfill supports use on automated liquid handling systems.

⁴KAPA UDI Primer Mixes and KAPA UDI Adapter plates are shipped with peelable seals. Replacement seals (three per plate) are peelable and pierceable.

*KAPA HyperPlex Adapters and KAPA UDI Adapters are not compatible with methyl-seq applications.

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Kit Size (reactions)	Roche Cat. No	Description	Kit Size (reactions)
KK8727	08861919702	KAPA Unique Dual-Indexed Adapter Kit (15 μM)	96 x 20 µL
KK8721	08278539001	KAPA Adapter Dilution Buffer	25 mL

KAPA UDI Adapters

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