Full-length Adapters

For Research Use Only. Not for use in diagnostic procedures.

Mitigate index mis-assignment with UDI strategies

The use of unique dual-indexing strategies can improve data quality by enabling the removal of reads containing unexpected barcode combinations, such as those caused by index hopping or index mis-assignment, prior to data analysis^{1, 2} (Figure 1).

Such index mis-assignment can occur during several steps of multiplexed sequencing, including library preparation (cross-contamination of barcodes or samples), amplification of pooled samples (template switching during PCR), and/or errors during sequencing and analysis—especially when patterned flow cells are used for sequencing.

Figure 1. Unique dual indexing mitigates the impact of index mis-assignment 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)².

Reduce the potential for index mis-assignment resulting from barcode cross-contamination with KAPA Adapters, which are manufactured using stringent procedures and then undergo sequencing-based QC testing.

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.

Save valuable time and resources

KAPA Adapters are a collection of high quality, unique dual-indexed adapters for ligation-based library construction. KAPA Adapters support many levels of sequencing multiplexing (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 Unique Dual-Indexed (UDI) Adapters, offering 96 adapters containing 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

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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 Adapters Selection Guide*

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	KAPA UDI Adapters	KAPA Universal Adapter
		KAPA Universal Adapter	KAPA Universal UMI Adapter
Number of UDIs (unique dual-indexes)	96	384
Number of 8-nt non-redundant P5 + P7 barcodes	96 + 96	384 + 384
Barcodes identical to those used in adapters supplied by Illumina	No(1)	No(1)
Recommended for all Illumina instruments with patterned or non-patterned flow cells	Yes	Yes
PCR-free library preparation	Yes	No
Molecular barcoded	No	No	No
Suitable for: KAPA HyperPrep & HyperPlus KAPA EvoPlus KAPA RNA HyperPrep KAPA HyperCap	Validated Compatible Validated Compatible(2)	Compatible Compatible Validated Validated	Compatible Compatible Validated
Compatible with KAPA Universal Enhancing Oligos	Yes	Yes
Adapter formulation	Full-length, ready-to-use, 15 μM 4 reactions/adapter(3)	Truncated, ready-to-use, 15 μM 1 reaction/adapter(3)	Truncated, ready-to-use, 33 μM 1 reaction/adapter(3)
Kit configuration	Hard-shell 96-well plate with automation-friendly labelling, overage and replacement seals(4)	Adapter tube and hard-shell 96-well primer mixes plates with automation-friendly labeling, overage and replacement seals(4)
KAPA Adapter Dilution Buffer	Yes (25 mL per kit)	No

⁽¹⁾The 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.

⁽²⁾ Theoretically possible but not fully tested.

⁽³⁾This 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.

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

* KAPA Universal Adapters, KAPA Universal UMI Adapters and KAPA UDI Adapters are not compatible with methyl-seq applications.

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KAPA Code	Roche Cat. No	Description	Kit Size	How to buy
KK8721	08278539001	KAPA Adapter Dilution Buffer (25 mL)	25 mL	Login for pricing
KK8727	08861919702	KAPA Unique Dual-Indexed Adapter Kit, (15 µM)	96 adapters x 20 µl each	Login for pricing

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KAPA Code	Roche Cat. No	Description	Kit Size	How to buy
09134336001	09134336001	KAPA UDI Primer Mix, 1-96	96 rxn	Login for pricing
09063781001	09063781001	KAPA Universal Adapter, 15 µM	960 µL	Login for pricing
09063790001	09063790001	KAPA Universal Adapter, 15 µM	4 x 960 µL	Login for pricing
09329862001	09329862001	KAPA Universal UMI Adapter, 33 µM	960 µl	Login for pricing
09329846001	09329846001	KAPA UDI Primer Mix, 193-288	96 rxn	Login for pricing
09329854001	09329854001	KAPA UDI Primer Mix, 289-384	96 rxn	Login for pricing
09329838001	09329838001	KAPA UDI Primer Mix, 97-192	96 rxn	Login for pricing

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Full-length Adapters

KAPA UDI Full-length adapters for PCR and PCR-free WGS, WES, and targeted sequencing workflows

Reduce the potential for index mis-assignment resulting from barcode cross-contamination with KAPA Adapters, which are manufactured using stringent procedures and then undergo sequencing-based QC testing.

Save valuable time and resources

KAPA Adapters Selection Guide*

KAPA Adapters Selection Guide*

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Full-length Adapters

Resources

Technical documents

Reduce the potential for index mis-assignment resulting from barcode cross-contamination with KAPA Adapters, which are manufactured using stringent procedures and then undergo sequencing-based QC testing.

Save valuable time and resources

KAPA Adapters Selection Guide*

KAPA Adapters Selection Guide*