Dilute Library to the Starting Concentration
This step dilutes libraries to the starting concentration for the NovaSeq 6000, NextSeq 500, NextSeq 550, NextSeq 1000, or NextSeq 2000 System. After diluting to the starting concentration, libraries are ready to be denatured and diluted to the final loading concentration.
Illumina recommends paired-end runs for sequencing. The number of cycles per Index Read is 10, and the number of cycles per read varies depending on the sequencing system.
| 1. | Obtain the molarity value of the library or pooled libraries using the applicable method: |
| • | For libraries quantified with a Bioanalyzer or TapeStation only, use the molarity value obtained for the library. |
| • | For libraries quantified with a Bioanalyzer or TapeStation, and Qubit, use the following formula to calculate the molarity value. Apply the average size from the Bioanalyzer or TapeStation, and the concentration from the Qubit. |
| • | For libraries quantified with a KAPA library quantification qPCR method, use the molarity value obtained for the library. Use 300–400 bp as the average library insert size. |
| 2. | Using the molarity value, calculate the volumes of RSB and library needed to dilute libraries to the starting concentration for your system. |
|
Sequencing System |
Starting Concentration (nM) |
Final Loading Concentration (pM) |
|---|---|---|
|
NextSeq 550 and NextSeq 500 |
|
1.1–1.4 |
|
NextSeq 1000 and NextSeq 2000 |
2 |
750 |
|
NovaSeq 6000 |
0.5 |
100 |
| 3. | Dilute each library to the starting concentration for your system using RSB. Combine 10 µl each diluted library in a tube to pool libraries. |
| 4. | Follow the denature and dilute instructions for your system to dilute libraries to the final loading concentration. |
If you are using the NextSeq 1000/2000 Control Software v1.2 or above, and not performing dark cycle sequencing (using the recommended alternate sequencing recipe without imaging for the first cycle of each insert read), trim the T-overhang and include at least 5% PhiX. Refer to the NextSeq 1000/2000 Product Documentation for more information on dark cycle sequencing.
