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适用说明:本说明书适用于KAPA Adapter试剂盒 (KK8330-8331-8332-8333)。
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Product Description

KAPA Adapter Kits for Ion Torrent platforms are designed for use with KAPA Library Preparation Kits for Ion Torrent platforms. Kits contain Adapter P1 in combination with either the non-barcoded Adapter A (KK8330), or a set of eight barcoded A adapters (KK8331 – KK8333). Libraries prepared using these adapters are suitable for sequencing on the Ion Personal Genome Machine™ and Ion Proton™ semiconductor sequencers.

Barcoded adapters allow for pooling of multiple fragment libraries prior to emulsion PCR (emPCR) in order to conduct multiplexed sequencing on a single chip.This simplifies the next generation sequencing workflow for a wide range of applications. Multiplexed sequencing also reduces the costs associated with emPCR, enrichment, and sequencing.

The sequences of the DNA barcodes in the barcoded KAPA Adapter Kits are identical to the sequences of the equivalent Ion Xpress barcodes and are thus optimized for equal representation of all barcodes in a pool. De-multiplexing of sequencing data is performed automatically by the Ion Torrent software. The highly dissimilar sequences of the barcodes minimize the possibility of incorrectly calling the barcode of a read due to sequencing or base calling errors.

Product Applications

KAPA Adapter Kits for Ion Torrent platforms are intended for use with the KAPA Library Preparation Kits for Ion Torrent platforms to generate libraries for either standard or multiplexed sequencing. Applications include whole genome shotgun sequencing, targeted sequencing by solution hybrid selection, RNA-seq, ChIP-seq, and amplicon sequencing.

Important Parameters

Adapter Concentrations
• The recommended adapter concentration is dependent on the amount of input DNA and the median fragment size of the library. As a general guideline, an adapter:insert molar ratio of between 10:1 and 20:1 is recommended.
• The recommended adapter concentrations for 130, 260, 320, and 410 bp inserts prepared from 100 ng – 1 µg of input DNA are provided in Table 1.
• KAPA Adapters are supplied at a concentration of 10 µM. When 10 µL of each adapter is used per 70 µL Ligation and Nick Repair reaction, the final concentration of each adapter is 1.4 µM.
• If a lower final concentration is required, a dilution of the 10 µM adapters to the appropriate concentration is recommended, such that addition of 10 µL of each diluted adapter to a 70 µL Ligation and Nick Repair reaction will result in the recommended final adapter concentration, as shown in Table 1.
• While it is not necessary to adjust adapter concentrations to accommodate moderate sample- to-sample variation in input DNA quantity, using an adapter concentration that is appropriate for the molar concentration of input DNA is recommended.
• It is important to maintain an adapter:insert ratio of ≥10:1 in order to minimize the formation of chimeric library inserts. Conversely, adapter:insert ratios higher than 20:1 may lead to reduced library yields.

Table 1. Recommended adapter concentrations (10 µL of stock per 70 µL Ligation and Nick Repair reaction)
 


Multiplexed Sequencing of Barcoded Libraries
Before pooling barcoded libraries for multiplexed sequencing, the concentration of each barcoded library should be determined accurately using qPCR (KAPA Library Quantification Kit for Ion Torrent), fluorometry or electrophoresis (e.g., Bioanalyzer).
After quantification, prepare an equimolar pool of barcoded libraries. Preparation of an equimolar pool can be achieved by first normalizing the individual libraries to the same concentration—before pooling equal volumes of each library.
Quantify the final library pool prior to template preparation using qPCR (KAPA Library Quantification Kit for Ion Torrent), fluorometry or electrophoresis (e.g., Bioanalyzer).

Adapter Sequences
Adapter P1
5' CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT 3'
3' T*T*GGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTA 5'

Adapter A
5' CCATCTCATCCCTGCGTGTCTCCGACTCAG 3'
3' T*T*GGTAGAGTAGGGACGCACAGAGGCTGAGTC 5'

Adapter A Barcode 1
5' CCATCTCATCCCT*G*CGTGTCTCCGACTCAGCTAAGGTAACGAT 3'
3' C-GCACAGAGGCTGAGTCGATTCCATTGCTA 5'
* phosphorothioate bond
- sequence alignment gap

The underlined portion of Adapter A Barcode 1 represents the position of the barcode, the sequence of which will vary as shown in Table 2. Aside from the unique barcode sequence, barcoded A adapters are identical.
Sequences of libraries produced with the barcoded adapter begin with the sequence TCAG[Barcode]GAT, followed by the insert sequence. The sequences of barcodes 1 – 24 are provided in Table 2.


Table 2. Barcode sequences
 

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