How to prepare low concentration (< 2 nM) libraries for sequencing on a MiSeq

The MiSeq Illumina Denature and Dilute Protocol Generator has directions for denaturing 2 nM and 4 nM libraries with the MiSeq Standard Normalization Method.

• For denaturation steps, make sure that the concentration of NaOH is no more than 1 mM in the final solution diluted with HT1. Higher NaOH concentrations can inhibit library hybridization to the flow cell, leading to decreased cluster density.

Libraries that are less than 2 nM but at least 0.5 nM can be prepared for MiSeq sequencing. This is done by following the Standard Normalization Method in the Denature and Dilute Libraries for the NextSeq 500 and NextSeq 550 Sequencing Systems Protocol Generator until libraries are diluted to 20 pM. In this protocol, excess NaOH is offset by adding 200 mM Tris-HCl, pH 7 to make sure that NaOH is fully hydrolyzed in the final solution.

• To denature < 2 nM libraries, follow the steps for starting library concentration of 1 nM or 0.5 nM in the Denature and Dilute Libraries for the NextSeq 500 and NextSeq 550 Sequencing Systems, see Figure 1. When libraries are at 20 pM, proceed to the Dilute Denatured 20 pM Library step in the Denature and Dilute Libraries for the MiSeq system under the section "Denature and Dilute, 4 nM Libraries", see Figure 2.

  

Figure 1.Denaturation and Dilution steps for Standard Normalization Method in the NextSeq 500/550 denature and dilution workflow.

Figure 2.Dilute Denatured 20 pM Library step for the Standard Normalization Method in the Denature and Dilute Libraries for the MiSeq system. To add a PhiX control, proceed to the "Denature and Dilute PhiX Control (Optional)" section of the Denature and Dilute Libraries for the MiSeq system.

Refer to the specific Denature and Dilute Protocol Generator for specific instruments for detailed instructions.

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