Why sequencing 25 or more cycles in Read 1 is recommended


The following guidance maximizes data yield and quality for customers sequencing custom libraries. All Illumina libraries are designed to be sequenced with a minimum first read length of 25 cycles. Illumina also recommends designing custom libraries to be sequenced with a minimum of 25 cycles to ensure reliable base calling and quality scoring.  

If specified during run setup, Illumina sequencing systems can perform fewer than 25 cycles for Read 1. This option is not recommended nor supported. Illumina primary analysis software, Real-Time Analysis (RTA), is optimized and validated to utilize the first 25 cycles of Read 1 for quality metrics calculations. For runs with fewer than 25 cycles in Read 1, RTA reports pass filter percentages and calculated quality scores from the available Read 1 cycles. However, as RTA performance has not been tested under these conditions, the selection of reads passing the chastity filter and the quality scoring metrics reported for these runs is not equivalent or directly comparable to those of runs with a Read 1 length of 25 or more cycles. For customers sequencing fewer than 25 cycles in Read 1, independent validation of the quality metrics is required.

For custom libraries with regions of low-diversity sequences such as internal barcodes, primer annealing sites, or homopolymers, consider increasing base diversity with the addition of a well-balanced library such as PhiX instead of sequencing a first read with fewer than 25 cycles. The percentage of PhiX to include with a custom library is instrument- and library-dependent and may require optimization. When sequencing libraries that use custom sequencing primers, utilize PhiX by spiking custom primers into Illumina primers. The following support bulletin provides additional information: Spiking custom primers into the Illumina sequencing primers.