• Over-amplification: During the late PCR cycles, primers are typically depleted before dNTPs, and excessive cycles may lead to accumulation of non-specific amplification products. These products migrate more slowly in electrophoresis and appear as diffuse high-molecular-weight bands. They still bind to the flow cell and sequence normally, so their presence does not significantly affect sequencing results. 
• Low sample purity: High concentrations of EDTA or elevated pH in the nucleic acid samples can inhibit DNA enzymatic digestion, resulting in incomplete fragmentation. It is recommended to purify nucleic acid samples according to Step 7: Post-amplification Cleanup using 2X NadPrep SP Beads and elute in Nuclease Free Water.

• Low input amount: When the total nucleic acid input is less than 5 ng, adapter dimers can form during library preparation, resulting in a higher proportion of shorter size distribution and lack of a clear main peak. Recommended solutions based on downstream applications:
I. Metagenomic sequencing (mNGS): When the total nucleic acid input is between 100 pg and 5 ng, it is recommended to purify the final library according to Step 7: Post-amplification Cleanup using 0.8X NadPrep SP Beads before sequencing.
II. Targeted capture sequencing (tNGS): Proceed directly to the liquid-phase hybrid capture workflow without size selection.

NadPrep ES Hybrid Capture Reagents v2 is compatible with the bead-based concentration (BC) method. Compared with the vacuum concentration (VC) method, BC method is simpler to operate, faster, and more suitable for automated platforms (see the Appendix of the user manual for details). It should be noted that due to differences in purification recovery efficiency, the captured library yield may be slightly lower when using BC method compared with VC method.

Cell loss during isolation: if cells are lost during isolation or transfer, inspect and optimize the cell isolation and collection workflow. Recollect samples and confirm that each reaction tube contains a cell before proceeding.
Insufficient purity of gDNA: residual wash buffers, ethanol or other contaminants remaining after purification can inhibit the amplification reaction. Ensure ethanol is fully evaporated after purification, resuspend/dilute purified gDNA in Nuclease Free Water, and verify accurate quantification of the input samples.

I. If the initial input samples are cells: apoptosis of cells prior to lysis can lead to DNA degradation, which may produce allelic loss or chromosomal abnormalities. It is recommended to assess cell viability and proceed with lysis and amplification immediately when the cell status is acceptable. If immediate amplification is not possible, store lysed cells at -25 ~ -15℃ for ≤ 7 days to preserve genomic integrity.
II. If the initial input samples are bulk DNA: partial degradation of the input gDNA can likewise cause such artifacts. Use intact, non-degraded gDNA, or increase the initial input amount as appropriate.

Exogenous DNA contamination: this kit is highly sensitive to trace DNA; ultra-low amounts of contaminating DNA can yield microgram-level amplification products. Perform all procedures in a clean, DNA-free environment (e.g., PCR-clean area or laminar-flow hood), and use nuclease-free, DNA-free consumables and reagents. Thoroughly decontaminate work surfaces, pipettes and equipment, and include and closely monitor negative controls throughout the workflow to rapidly identify and trace contamination sources.

Yes. NadPrep EZ RNA Library Preparation Kit is compatible with a variety of rRNA-depletion and mRNA enrichment modules, and it can be combined with targeted capture workflows. Recommendations:

1)      rRNA depletion (recommended): Preferentially use the species-appropriate NadPrep rRNA Blocking Reagent and follow Appendix 2: Connect to Upstream Modules [Connect to NadPrep rRNA Blocking Reagent (Recommended)] to complete the library preparation. The following products are available for selection:

• NadPrep rRNA Blocking Reagent (Human) (Cat #1002901/#1002902)

• NadPrep rRNA Blocking Reagent (Zebrafish) (Cat #1002911/#1002912)

• NadPrep rRNA Blocking Reagent (HMR) (Cat #1002921/#1002922)

•  NadPrep rRNA Blocking Reagent (Plant) (Cat #1002931/#1002932)

• NadPrep rRNA Blocking Reagent (Custom) (Cat #10029XX)

2)      mRNA enrichment: Third-party mRNA enrichment modules may be used flexibly. Total RNA processed by the corresponding procedures should be eluted in Nuclease Free Water, and then processed with Step 1: RNA Fragmentation & Random Primers Annealing for library preparation in the user manual.

3)      Targeted RNA-Seq: It is recommended to connect with the Hybrid Capture Systems from Nanodigmbio for target enrichment. And complete the captured-library preparation according to the corresponding user manual (www.njnad.com).

For high-integrity RNA samples from cells or fresh tissues (Grade A/B), Scheme I: Size Selection is recommended to obtain an optimal size distribution in the final library.

For low-integrity RNA samples from highly degraded cells or frozen tissues (Grade D) or FFPE-derived RNA (Grade C/E), Scheme II: Purification is recommended. Because these samples are already highly fragmented, directly purifying the ligation products helps retain original sequence information and minimizes loss of effective reads.

• RNase contamination: Use RNase-free consumables throughout the workflow and perform all the operations in RNase-free conditions. RNase contamination will reduce reverse transcription efficiency and low library yield.
• Low sample purity: Residual contaminants in RNA (e.g. chelators, guanidine salts, phenol, ethanol) may inhibit the activity of reverse transcriptase. It is recommended to purify RNA according to Step 7: Post-amplification Cleanup using 2X NadPrep SP Beads and elute in Nuclease Free Water.
• Poor sample quality: For low-quality RNA samples, follow the recommendations to increase the no. of PCR cycles appropriately.

Yes. It is. NadPrep EZ RNA Library Preparation Kit is suitable to most RNA samples from different grades and sources (cells, fresh tissues etc.). For FFPE-derived RNA samples with severe fragmentation (DV₂₀₀ < 20), reverse transcription efficiency may be reduced, and it is recommended to increase the no. of PCR cycles as appropriate. When performed targeted capture on FFPE-derived RNA samples, consider increasing the RNA input to improve capture efficiency.