Improving Illumina Sequencing Performance

Example 4

To evaluate the effect of an added antioxidant in the ScanAndFill mix (no polymerase) on detection step signal decay and sequencing error rate, a polynucleotide of a known sequence was sequenced using an Illumina MiniSeq™ sequencer using, during the detection step, a ScanAndFill mix without a polymerase and with or without 3 mM ascorbate. The ScanAndFill mixes included EA buffer, pH 9.85, MgSO₄, EDTA, A-LN3, C-LN3, T-LN3, Dark G, and CHAPS. 100 cycles of sequencing were run using incorporation times of 7.5 seconds and were compared to a baseline using the standard scan mix with an incorporation time of 25 seconds per cycle. The results are shown in FIGS. 8-9.

The addition of ascorbate resulted in substantially improved signal decay reduction (FIG. 8) and reduction in error rate (FIG. 9) relative to the ScanAndFill without ascorbate. Read lengths of greater than 75 cycles are enabled with the addition of the antioxidant. As shown in the FIG. 9 inset, ascorbate also improved the % Q30. Additionally, ascorbate improved the % Align PhiX.

Similar results were observed with other concentrations of ascorbate (up to 20 mM), with varying concentrations of acetovanillone (up to 20 mM), and combinations of ascorbate and acetovanillone (data not shown).

In addition, the pH may be varied, and similar results may be obtained.

A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made. Accordingly, other embodiments are within the scope of the following claims.