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Sample Multiplexing Calculator
Determine the number of samples you can multiplex in a sequencing run for your QIAseq NGS workflow
Sample Multiplexing Calculator
This calculator helps you determine the approximate number of samples that can be multiplexed on your sequencing instrument to reach the required coverage for your QIAseq panel. Before starting a sequencing experiment, you should know the coverage (depth) of sequencing you need to achieve. Please refer to “How do I calculate mean coverage?” to determine the coverage you need to achieve for your panel.
Insights
Our Sample Multiplexing Calculator is a vital tool for researchers and laboratory technicians who are preparing for next-generation sequencing (NGS) experiments. This calculator aids in determining the optimal number of samples that can be multiplexed on a sequencing instrument, ensuring that you achieve the necessary coverage for your QIAseq panel.
How It Works
- Input your data: Begin by entering the specific details of your sequencing panel, including the recommended UMI coverage and the amount of DNA input.
- Receive your estimate: The calculator will provide an estimated number of samples that can be multiplexed to reach the desired sequencing depth.
Importance of Accurate Quantification
Accurate quantification of libraries using qPCR is crucial before pooling, as it impacts the clustering efficiency on the flow cell and the evenness of read distribution among samples. Inaccurate quantification can lead to suboptimal results, affecting the overall quality of the sequencing data.
Calculating Mean Coverage
Several variables must be considered to determine the required coverage depth:
- Sequencing platform: Different platforms may have varying efficiency and output, influencing how many reads are necessary for sufficient coverage.
- Sequence complexity: More complex regions may require deeper coverage to ensure all variants are captured.
- Sample type and input: The quality and quantity of the starting material can affect the number of reads needed.
- Variant types: The nature of the variants being studied (e.g., SNVs, indels) influences the depth required for reliable detection.
DNA Input Amount and Coverage Depth
The calculator also takes into account the correlation between the DNA input amount and the sequencing depth. It provides guidance on the adequate depth of coverage required to sequence captured UMIs effectively.
Variant Detection
For sensitive detection of somatic variants, particularly those with a Variant Allele Frequency (VAF) below 1%, the calculator can guide you in adjusting the DNA input and sequencing depth. This ensures that a sufficient amount of UMIs are generated, increasing the likelihood of detecting these low-frequency variants.