TITLE: Appendix 2: Optimization of Fragmentation Parameters
AUTHORS: Dakota Betz

[DESCRIPTION]
Fragmentation guidelines provided in the Library Construction Protocol: Enzymatic Fragmentation (step 1) may not result in the optimal library size distribution for your specific DNA samples. For this reason, precious samples should not be used when evaluating the KAPA HyperPlus Kit for the first time. Instead, fragmentation parameters should be optimized in the context of the KAPA HyperPlus workflow, using a non-precious, bulk DNA sample that is representative of the  actual samples to be processed. 

The information in this Appendix should be considered during the experimental design for your evaluation of the KAPA HyperPlus Kit.

[BEFORE_START]
Shipping and Storage
The enzymes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and
storage. KAPA HyperPlus Kits are shipped on dry ice or ice packs, depending upon country of destination. Upon
receipt, immediately store enzymes and reaction buffers at -15°C to -25°C in a constant-temperature freezer. When
stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date
indicated on the kit label.

Handling
Always ensure that KAPA HyperPlus Kit components have been fully thawed and thoroughly mixed before use.
The End Repair & A-Tailing Buffer and Ligation Buffer may contain precipitates when thawed at 2°C to 8°C. These
buffers must be thawed at room temperature and vortexed thoroughly before use. KAPA HiFi HotStart ReadyMix (2X)
contains isostabilizers and may not freeze completely, even when stored at -15°C to -25°C. Nevertheless, always
ensure that the ReadyMix is fully thawed and thoroughly mixed before use. Reaction master mixes prepared from
the enzymes and buffers for fragmentation, end repair and A-tailing, as well as for ligation, are very viscous and
require special attention during pipetting. Keep all enzyme components and master mixes on ice as long as possible
during handling and preparation.

Quality Control
All kit components are subjected to stringent functional quality control, are free of detectable contaminating exoand
endonuclease activities, and meet strict requirements with respect to DNA contamination.

[STEPS]
1. 
 

2. Quantification of Input DNA

The Qubit fluorometer is recommended for the quantification of high-quality DNA, whereas the
KAPA hgDNA Quantification and QC Kit provides both concentration and quality information for FFPE DNA.
3. Handling of DNA Samples Containing EDTA

 

4. For example:
• If your DNA samples are in TE buffer, and your input into library construction is 100 ng, dilute 100 ngof each sample into a final volume of 30 µL (i.e., to 3.33 ng/μL ) using TE buffer.

• All samples will now contain the same final EDTA concentration once diluted to 50 µL for fragmentation.
This concentration is: EDTA concentration in TE buffer x (30 μL/50 μL) = 1 mM x (30 μL/50 μL) = 0.6 millimolar (mM)
   
• Make a 2.2-fold dilution of the Conditioning Solution (as per Table 3 on p. 4), and follow the Library
Construction Protocol (second half of step 1).
5. 
 

5.1. Set up a series of test reactions with the appropriate amount of input DNA, and different final concentrations
of Conditioning Solution.
5.2. Include at least one reaction with the same input of control DNA known to be EDTA-free. The control DNA
should preferably be of the same type and quality as the test samples.
5.3. Fragment the DNA using the appropriate parameters, as outlined in the Library Construction Protocol:
Enzymatic Fragmentation (step 1). Complete the library construction process, and compare library size
distributions for the test and control samples using an electrophoretic system (see Important Parameters:
Evaluating the Success of Library Construction, p. 8).
5.4. Titrate the final concentration of Conditioning Solution in the reaction until the test samples yield similar
fragmentation profiles as the EDTA-free control sample, or until the desired library size distribution has been
achieved.
5.5. A two-step strategy may be the best. Start with 3 – 4 test samples covering a broad range of final Conditioning
Solution concentrations, then perform a finer titration over a narrower concentration range.