Frequently asked questions

Next Generation Sequencing

• When BiOligo performs annealing of NGS adapters for customers, what is the approximate annealing efficiency and final concentration? Can you also provide the dilution buffer?
  The annealing efficiency is not quantified, but it can be verified by agarose gel electrophoresis. For short adapters, the annealing efficiency is typically close to 100%. The final concentration can be customized upon request, with our standard concentration being 15 µM. We can also provide the annealing buffer for dilution purposes.
• What is the principle behind DNA sequencing? What are the steps? How accurate is ABI 3730XL sequencing?

  The principle of DNA sequencing is the Sanger method (also known as the chain termination method). The specific steps are as follows:

  
  

  1.  Quantification: Quantify the sample and primers.

  
  

  2.  Sequencing Reaction: Add the quantified template and primers to the reaction system along with reagents like BigDye, and perform the sequencing reaction using a PCR machine.

  
  

  3.  Data Reading: Based on the different fluorescent signals, read the base sequence of the tested sample. ABI (Applied Biosystems) currently claims an accuracy of 98.5% for sequencing results within 800 base pairs using the ABI 3730XL sequencing system.

  
  

• For the pre-made adapters, 5 μL is used per reaction. Should the same volume be used for low-input library preparation (e.g., 1 ng DNA input)?
  For DNA inputs ≥ 50 ng, 5 μL of adapter can be added directly per reaction. For inputs < 50 ng, the adapters should be diluted accordingly. For 1 ng DNA input, dilute the adapters 5- to 15-fold, then add 5 μL of the diluted adapter solution.
• What are the specifications of your UMI adapters? How many UMI bases are included, and what does the "S" represent in the adapter design?
  Our UMI adapters have demonstrated superior performance in benchmarking studies, offering: 1. Higher library yield and more effective UMI complexity compared to competitors. 2. Consistent UMI length across different platforms:   • UMI positions span bases 1 to 5 (N positions, starting from the first base).   • Bases 6 and 7 are not part of the UMI. 3. A unique adapter design: NNNN(S)T (7 bases in total).   • The "S" represents two specialized bases — a proprietary feature exclusive to BiOligo.

  
  

• Regarding your library prep kits (e.g., 96-reaction kits), can all 96 reactions be reliably performed? Some kits from other suppliers may have losses, resulting in only 92–93 usable reactions.
  Yes, our kits reliably deliver a full 96 reactions. BiOligo's design takes potential processing losses into account to ensure that all 96 reactions can be successfully completed.
• How many pmol of capture probes are used per reaction in the panel? How should the dry probe panel be diluted?
  Probe amount per reaction: 3 pmolNote: For smaller panels (<5,000 probes), this amount may be reduced to optimize costs.Dilution protocol for lyophilized probe panels:  ① Calculate total probe amount: Total pmol = Number of probes × 500 pmol  ② Resuspend in Low TE Buffer (pH 8.0) to a final concentration of 3 pmol/μL.   ‣ Example: A 10,000-probe panel      10,000 × 500 pmol = 5,000,000 pmol      Add 1,667 μL Low TE → final concentration = 3 pmol/μL  ③ Usage per reaction: Add 1 μL of the diluted probe solution (delivering 3 pmol)

  
  

• Is the amount of blocker added different between multiplexed and singleplexed reactions? How many pmol or μg are used per reaction?
  The amount of blocker used is the same for both multiplexed and singleplexed workflows.Per reaction usage:Volume: 2 μL Molar amount: 2 nmol Mass: 66 μg (based on the blocker’s molecular weight)Rationale: Blockers are added in excess to ensure complete suppression of nonspecific binding, regardless of the level of sample multiplexing.
• Many library prep kits—especially those used for pathogen detection—emphasize being "free of background microbial contamination." Is this solely due to the cleanliness of the production facility?
  Not entirely. While our facility operates under strict contamination control in GMP-grade laboratories, the "microbe-free" status is achieved through multiple factors:  • Ultra-low levels of host nucleic acid residues in enzyme raw materials  • In-house enzyme production with stringent residual DNA monitoring  • Endotoxin-free manufacturing pipelines
• Are BiOligo's capture probes single-stranded (ssDNA) or double-stranded (dsDNA)? What are the advantages of single-stranded probes compared to double-stranded ones?
  BiOligo primarily uses single-stranded DNA (ssDNA) probes. Compared to double-stranded (dsDNA) probes, ssDNA offers several key advantages:

  • Higher quality: Each probe is synthesized individually via column synthesis, allowing for per-probe quality control (MS/HPLC), achieving >98% purity, versus 90–95% for chip-synthesized dsDNA.

  • Superior uniformity: Precise 1:1 molar mixing ensures >95% probe consistency, whereas chip-synthesized dsDNA often shows ≥10% yield variation.

  • Greater yield: Column synthesis yields ≥50 nmol per probe, compared to ≤1 nmol per probe from chip synthesis.

  • Best-use scenarios:

    1. ssDNA is ideal for panels with fewer than 5,000 probes, where high accuracy is critical.

    2. dsDNA is more cost-effective for large panels with more than 10,000 probes.