NEBioCalculator^® qPCR NGS Library Quantitation Module

In this video I will demonstrate how to use NEB’s online NEBiocalculator^® tool to determine the concentration of Next Generation Sequencing (NGS) libraries that have been quantitated using qPCR. 

To access the NEBiocalculator tool go to: https://nebiocalculator.neb.com.  You can also find it on the neb.com website, under tools and resources, calculators, NEBiocalculator.  Once in the NEBiocalculator page locate the qPCR heading, and click on the NGS Library Quant module section.  You are now on the qPCR NGS Library quantitation tool page.

To use the tool, first select the kit that was used to quantitate your libraries.  You can select between the NEBNext^® Library Quant Kit for Illumina or the NEBNext Library Quant Kit for Ultima Genomics.  Note that it is important to select the appropriate kit, as the standards for each kit are different sizes and this will influence the calculation of the library concentrations.  If you wish to learn more about these products, click on the product name and it will direct you to the product page.  For this video, we will select the NEBNext Library Quant kit for Ultima Genomics as an example.  

To upload your data, use the template provided.  The template can be easily accessed by clicking “download template”.  Note that clicking the file format link will display an example of the completed template.  Once in the template, enter the quant values for the standards.  Quant values for up to three replicates can be entered.  Note that quant values can be reported as Cq (quantification cycle) or Ct (threshold cycle), depending on the qPCR instrument.  Either will work, as they are both treated the same by the tool. For simplicity we use Cq.  The standards are listed from lowest to highest concentration.  If more convenient, you can reorder the rows from highest to lowest without affecting the calculation.   

Next, you will add the library information.  To enter your library id, replace the text that reads “Example Library 1” with your desired library or sample name. For example, this library is called: "My test library 1”.  It’s important to keep the pound sign “# “at the beginning of the field as this indicates to the tool that this row contains the library name.  On the row that reads dilution, enter your library dilution, this library I diluted 4000-fold.  Also enter the quant values, up to three replicates, and average library size.  It’s important to enter the library size as this value will be used to calculate the concentration. If no value is entered, the default value of 200 bp (which is the size of the standard), will be used.  

For multiple dilutions, add each dilution in a row under the library name. Feel free to add rows if needed.  Here I will add a 10,000 fold dilution for “My test library 1”.  To add a new library, start a row using the pound “#” sign, followed by the library name. In this case, is “My test library 2”.  In the row below, add the Dilution id and proceed to fill out the information as done with the previous library.  Feel free to add rows to add more dilutions or libraries if needed.  Once you have entered all your library information, save your file in csv format. 

You can then upload your data by pressing “Select File” and selecting your saved csv file.  If you need to remove the selected file, you can do so by pressing  “clear file".  Once the data is uploaded, the tool will display the results in the web interface. 

For the standards, the tool will calculate the average Cq values among replicates and generate a standard curve.  The average Cq values are plotted on the Y-axis against the log concentration of the standards on the X-axis.  It will display the best-fit line, and report the line’s y intercept, slope, and R2 value.  It will also report the qPCR efficiency, a measure of product duplication at every amplification cycle.  A 100% efficiency indicates doubling of the target amplicon at each PCR cycle. 

For the libraries, the tool will calculate the Average Cq and the concentration of the undiluted library.  All dilutions are considered by the tool when calculating the Average Concentration of the Undiluted library.  The concentration of your undiluted libraries is reported in nM.

If using the tool interphase, first enter the quant values for the standards in the provided spaces. Values for up to three replicates can be entered.  Note that quant values can be reported as Cq (quantification cycle) or Ct (threshold cycle), depending on the qPCR instrument.  Either will work, as they are both treated the same by the tool. For simplicity we Cq.

Once the values are entered, the tool will calculate the average Cq values among replicates and generate a standard curve. The average Cq values are plotted on the Y-axis against the log concentration of the standards on the X-axis.  It will display the best-fit line, and report the line’s y intercept, slope, and R² value.  It will also report the qPCR efficiency, a measure of product duplication at every amplification cycle. A 100% efficiency indicates doubling of the target amplicon at each PCR cycle.  Note that if its more convenient, the standards can be reordered, highest concentration to lowest using the sort order button. Make sure you do this before entering the Cq values.

The second step is to enter your library information.  To do this press “+ Add Library".  Enter your Library name, the library dilution, for example our first dilution is a 1:4000 dilution, the Cq values for your replicates, and fragment size.  It’s important to enter the library size as this value will be used to calculate the concentration. If no value is entered, the default value (displayed), will be used. 

The tool will calculate the Average Cq and the concentration of the undiluted library.  If you have multiple dilutions of this library, press “Add a dilution” to enter the information.  All dilutions are considered by the tool when calculating the Average Concentration of the Undiluted library.  To remove a library from the calculation, press “remove” next to the unwanted library.  The concentration of your undiluted library is reported in nM.  For multiple libraries, “press Add a library” and enter the information as done with the previous library.

The final step is to save your results.  You can save the results in an editable csv format or download a report containing the input data and results, in pdf format.  For details on the formula used to calculate the library concentration, refer to the “formula” information section at the bottom of the page.

If you have additional questions, contact NEB technical support.  Thank you for watching.