Product Description
KAPA Single-Indexed Adapter Kits for Illumina platforms are designed for use with KAPA DNA and RNA library preparation kits to construct libraries for sequencing on an Illumina sequencer. Kits contain a set of 12 or 24 adapters, each with a single, 6-nucleotide index (barcode) for multiplexed sequencing applications.
Adapters are available in high (30 μM) or low (1.5 μM) concentration kits. Please refer to Table 1 in Important Parameters: Compatibility with KAPA Library Preparation
Kits (p. 3) for guidelines on which concentration to use for different library types.
The sequences of the sequencing indices (barcodes) included in KAPA Single-Indexed Adapter Kits are given in Important Parameters: Index Sequences and Pooling Guidelines (p. 6). The highly dissimilar nature of indices minimizes the possibility of inaccurate index calling.
Adapter Dilution Buffer (10 mM Tris-HCl, (pH 8.0-8.5), 10 mM NaCl, 1 mM EDTA) is provided with the kit to ensure optimal performance when adapters have to be diluted.
Product Applications
KAPA Single-Indexed Adapters are used to uniquely label sequencing libraries generated from individual biological samples. This allows for the pooling of libraries prior to target capture or cluster generation, to enable multiplexed sequencing – which simplifies sample preparation and reduces the cost of next-generation sequencing for a wide range of applications. Primary applications for the use of KAPA Single-Indexed Adapter kits for Illumina platforms include:
•whole exome or targeted sequencing, using Roche SeqCap EZ or IDT xGen Lockdown Probes or other hybridization capture systems, in combination with the appropriate blockers
•RNA-seq
•ChIP-seq
•other direct sequencing applications, e.g., microbial whole-genome sequencing on compatible platforms.
Note: KAPA Single-Indexed Adapter Kits are not validated for sequencing on the Illumina HiSeq X instrument. In addition, KAPA Single-Indexed Adapters are not methylated, and can therefore not be used for methyl-seq applications.
Important Parameters
Best Practices
•KAPA Single-Indexed Adapters should be used on ice or in cooled reagent blocks, and must not be exposed to conditions above room temperature.
•Use the Adapter Dilution Buffer (10 mM Tris-HCl, (pH 8.0-8.5), 10 mM NaCl, 1 mM EDTA) provided in the kit to dilute KAPA Single-Indexed Adapters. Adapters diluted in any other buffer or in PCR-grade water may not support optimal library construction efficiency.
•Dilute only the amount of each adapter needed for same-day usage. Long-term storage and multiple cycles of freezing and thawing of diluted adapter stocks are not recommended.
•Employ best laboratory practices to avoid crosscontamination of adapters and/or the dilution buffer.
•Always use plastics that are certified to be free of DNases, RNases, and nucleases. Low DNA- and RNAbinding plastics are highly recommended, especially for low-input DNA and all RNA-Seq library construction applications.
Compatibility with KAPA Library Preparation Kits
KAPA Single-Indexed Adapter Kits for Illumina platforms are recommended for use with all KAPA library construction kits.
Table 1 indicates which adapter stock concentration (30 μM or 1.5 μM) is best suited for library construction depending on the input amount and the kit used for library preparation.
Table 1. Recommended adapter concentration by input
*Please consult individual Technical Data Sheets for RNA HyperPrep Kits for input recommendations.
** For 100 ng input, the recommended adapter concentration depends on average insert size.
When selecting a KAPA Single-Indexed Adapter Kit for your application, please consider the following:
•Refer to the table corresponding to the KAPA library preparation kit that you will be using. Recommended adapter stock concentrations for libraries constructed from different inputs and DNA fragment lengths with the kits listed above are included in Tables 2 – 6 on p. 4.
•Identify the recommended adapter stock concentration for your input and fragment length. If that concentration is > 1.5 µM, use a high concentration (30 µM) KAPA Single-Indexed Adapter Kit. If that concentration is ≤ 1.5 µM, use a low concentration (1.5 µM) KAPA Single-Indexed Adapter Kit.
•If your input and specific fragment length is not listed in the table, follow the instructions in Adapter Concentration Calculations (p. 5) to calculate the appropriate adapter stock concentration for your experiment, or contact sequencing.roche.com/support to obtain a calculator designed for this purpose.
•For each batch of libraries to be constructed, prepare an appropriate volume of diluted adapter using the Adapter Dilution Buffer provided in the kit, remembering that:
- Standard protocols call for 5 µL of appropriately diluted adapter stock per library.
- If an adapter stock concentration > 30 µM is required, the volume of water in the ligation reaction may be reduced and the volume of adapter increased to the same extent, up to a total of 10 µL adapter per reaction.
- Diluted adapters should be freshly prepared and must not be stored for long periods of time or subjected to repeated freezing and thawing.
- An excess volume of each diluted adapter stock will be required to ensure accurate dispensing. The excess will be larger for automated vs. manual use.
- All or some of the libraries in the batch may require a different index. Please refer to Index Sequences and Pooling Guidelines (p. 6) for recommendations on multiplexing.
- The Technical Data Sheet included with your library preparation kit contains specific guidelines for the optimization of adapter concentration when using that particular kit for different applications.
Table 2. Recommended adapter concentrations for KAPA Hyper Prep and HyperPlus Kits1
1Adapter: insert molar ratio calculations are based on a mode DNA fragment length of 200 bp, and will be higher for longer DNA fragments, or slightly lower for DNA fragmented to a mode size < 200 bp. The lower adapter: insert molar ratios recommended for inputs > 100 ng represent a fair compromise between library construction efficiency and cost; higher library yields will be achieved if a higher adapter concentration is used.
Table 3. Recommended adapter concentrations for KAPA HTP and LTP Library Preparation Kits1
1Adapter concentrations are based on a ~10:1 adapter:insert ratio. For values not listed here, please refer to the calculation in Important Parameters: Adapter Concentration Calculations or visit kapabiosystems.com/support to obtain a calculator designed for this purpose.
Table 4. Recommended adapter concentrations for KAPA RNA HyperPrep Kits
Table 5. Recommended adapter concentrations for KAPA Stranded RNA-Seq Kits with RiboErase (HMR)
Table 6. Recommended adapter concentrations for KAPA Stranded RNA and mRNA-Seq Library Preparation Kits
