Size Selection	Applicable Situation	Advantage	Disadvantage
Step 1 Optional steps: Double-sided size selection after fragmentation	Sufficient DNA input; Broad size distribution of DNA fragments after fragmentation	Narrow size distribution, high ligation efficiency and uniform sequencing data	Loss of sample
Step 4 Optional steps: Double-sided size selection after ligation	Sufficient DNA input; Broad size distribution of libraries after adapter ligation	Narrow size distribution, Uniform sequencing data	Strict double-sized selection parameters based on library size

• Loss caused by double-sided selection after fragmentation

Experimental conditions: 500 ng of Human Genomic DNA Male (Promega, catalog # G1471) was fragmented by using different enzymatic digestion times, and the procedures were conduct at 25℃ for 15, 20, 25 and 30min, respectively, then65℃ for 30 min. At the end of the fragmentation procedures, according to Appendix II 1.1 of the user manual, double-sided selection was used. After selection, Nuclease Free water was used to elute for subsequent adapter ligation.

This step of selection can make the fragmented samples more concentrated, thus leading to high efficiency of subsequent adapter ligation, but the sample loss is relatively high (about 60-70%), which is suitable for the library preparation of sufficient DNA. It is recommended that input DNA is ≥200 ng

• Loss caused by double-sided selection after ligation

Experimental conditions: 100 ng of Human Genomic DNA Male (Promega, catalog # G1471) was fragmented by using different enzymatic digestion times. After the adapter ligation was

completed, the single screen mode and double screen mode were used respectively (see Appendix II 1.2 of the user manual for details).

The library yield after double-sided selection is 15-25% loss compared with  single selection. But it is not recommended for low-quality DNA or input DNA is ≤50 ng.

• Sample quality difference: Whether the samples are degraded. For degraded samples, the enzymatic digestion time can be shortened in a gradient of 3-5 min according to the recommended time in the user manual.
• Sample solvent: Solvent contains EDTA or pH is too high (pH greater than 8.0, such as pH 8.5). When the sample solvent contains EDTA, if it is not purified, Enhancer Buffer is not added, or the solvent pH is too high, it will lead to insufficient fragmentation.

A. 50 ng blood gDNA samples containing EDTA (0-0.8 mM) were used for library preparation. The higher the EDTA concentration, the larger the enzymatic digestion fragment.

NadPrep EZ DNA Library Preparation Kitv2 is suitable for various types of samples, including whole blood gDNA, FFPE, etc. For the treatment of complex samples (such as FFPE samples), it is recommended to carry out quality control at the source. If there are impurities such as protein and phenols in DNA samples, the effect of enzymatic digestion may be affected.

• Sample solvent: It is recommended to use nuclease Free Water or 10 mM Tris-HCl (pH 7.5-8.0) to dissolve the DNA samples (the enzymatic fragmentation reagent is sensitive to the concentration of EDTA. Therefore, it is necessary to confirm the concentration of EDTA in the solvent in this step. If the solvent pH is too high, the enzyme activity might be affected, resulting in insufficient fragmentation (1.8X magnetic bead purification can be carried out if necessary).
• Sample quantification: It is recommended to use NanoDrop based on the principle of spectrophotometer as well as Qubit® and PicoGreen® based on double-stranded DNA fluorescent dye for quantitative analysis of general gDNA samples.
• Sample purity: High quality DNA input, with OD260/OD280=1.8-2.0；OD260/OD230=2.0-2.5.
• Sample fragment distribution: It is recommended to use 1% agarose gel electrophoresis for sample size detection or to use bioanalyzer (such as Agilent 2100) DIN value for sample integrity assessment. The samples can be simply classified into four grades as shown in the following figure (the basic four grade classification method; samples can also be further classified according to the actual situation). The enzymatic digestion time of different grades of FFPE samples can be adjusted according to the user manual.

Grading Standard as follows:

A:One clear  strip with about 15 kb in size

B+:One clear and slight trailing strip with about 15 kb in size

B:One indistinct strip with about 15 kb Consistent withg DNA One extra cycle more in size, with medium diffusion

C:Multiple strips ranging from 200 bp to 2,500 bp, with severe diffusion

D:Multiple strips ranging from 100 bp to 1 kp, with severe diffusion

No, Nuclease-Free water shall be added to  a total volume of 40μL. The composition of TE Solution is 10 mM Tris, 0.1 mM EDTA (pH 8.0), which can be used for library storage.

The degree of fragmentation of FERA Enzyme depends on time and temperature. The size of DNA fragment depends on the enzymatic digestion time, and the size of DNA fragment can be flexibly adjusted according to the enzymatic digestion time.

The library for RNA-Seq can be prepared with this module coupled with the rRNA removal module. The NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB, Cat # E7400L) is recommended.

The RIN of RNA extracted form FFPE tissue is generally small, of which the value is mostly around 2.0. While the DV 200 differs greatly among the RNA samples, which indicates the actual degradation degree of RNA extracted from FFPE tissue. Therefore, we highly recommend DV 200 as the QC reference of RNA from FFPE tissue. 

This module is suitable for most of RNA samples from cells and tissues. As for the highly degraded RNA from FFPE tissues (DV 200 < 30%), the efficiency of dsDNA targeting declines greatly. When using RNA from FFPE tissues for targeted sequencing, it is recommended to augment the RNA input to improve the capture efficiency.

Make sure to proceed the RNA-related steps using RNase-free consumables in the area without RNase contamination. Furthermore, additional purification step by using NadPrep SP Beads is necessary for wiping off the residual impurities, which may inhibit the subsequent enzymatic reaction, including chelating agent, guanidine salt, phenol, ethanol, etc.