Sodium hydroxide (NaOH) is a critical reagent used to denature double-stranded libraries (Figure 1) before loading onto Illumina sequencing systems. This step is performed by the user, except for the onboard denaturation workflows for the iSeq 100 and NextSeq 1000/2000 systems. Complete denaturation results in single-stranded libraries, which can form clusters by hybridizing to the P5 and P7 oligos that are covalently attached to Illumina flow cell surfaces, as illustrated in Figure 2. Improper denaturation can result in decreased sequencing yields.

Figure 1: Double-stranded library fragment, before denaturation.

Figure 2: After complete denaturation, single-stranded library fragments bind to the flow cell by hybridization to the P5 oligo (shown) or P7 oligo that are covalently attached to the flow cell surface.

To denature libraries, it is critical for the concentration and pH of NaOH to be at recommended levels – typically 0.1-0.2 N NaOH and pH > 12.5, respectively. NaOH has a high affinity for atmospheric carbon dioxide (CO₂), which can acidify the reagent. When the pH drops below 12.5, incomplete denaturation can occur, which can result in lower cluster densities.

To prevent acidification of NaOH used for sequencing, Illumina recommends the following steps:

• Upon receiving stock NaOH (usually 1 N to 10 N), create ~1-2 ml aliquots of stock NaOH. Use screw cap tubes and seal with Parafilm (or equivalent). Store aliquots of stock NaOH at -20°C. Discard aliquots after one year if not consumed.
• When setting up a sequencing run, remove one stock aliquot from -20°C storage to thaw on the lab bench. This stock aliquot may remain on the bench at room temperature until consumed or up to one week, whichever happens first.
• Discard any unused stock aliquot after one week and remove a fresh aliquot for the next required usage.
• Dilutions of the stock NaOH to 0.1 or 0.2 N for library denaturation should be made fresh and used within 12 hours.
  • Discard any diluted 0.1N or 0.2N NaOH after 12 hours has elapsed.
  • Refer to the Denature and Dilute Libraries Guide for your instrument for detailed instructions.