Making sure that sufficient volumes of SBS reagents are loaded onto the sequencing instrument is crucial for a successful run. Running out of reagents during a run impacts intensity and quality, and can result in a run failure. Dual-index sequencing, depending on the instrument, follows different workflows, as shown in the Indexed Sequencing Overview Guide. On certain instruments, dual-index runs need an additional seven cycles of reagents for the chemistry-only cycles at the beginning of the second Index Read. The seven chemistry-only cycles are required for the workflows in which the second Index Read sequencing occurs after the forward template anneals to the grafted P5 primer on the surface of the flow cell. These seven cycles must be considered when calculating the number of cycles supported by the kit.
The following table outlines available reagent kit sizes and the maximum number of cycles each kit can perform, taking the additional seven cycles into consideration:
|Reagent Type||Reagent Kit Size||Maximum Number of Cycles||Additional Cycles Needed for Dual Index?|
|iSeq™ 100||i1 (v1 or v2)||300||322||No|
|MiniSeq™||Rapid Kit||100||128||Yes - 7 cycles|
|High Output or |
(including Micro and Nano kits)
|50||79||Yes - 7 cycles|
|NextSeq™ 500/550||High Output or Mid Output||75||92||No|
|HiSeq™ 1000/1500/2000/2500||Rapid SBS v2||50||79||7 cycles required for paired-end flow cells|
|TruSeq SBS v3||50||58|
|HiSeq SBS v4||50||79|
|HiSeq 3000/4000||N/A||50||79||7 cycles required for single-read flow cells|
|NovaSeq™ 6000||SP v1.0||100||130||Yes - 7 cycles|
|S1 and S2 v1.0||100||130|
|S1 and S2 v1.5||100||138|
*The NextSeq 1000/2000 P2 v3 kit has 338 total cycles while the P3 v3 kit has 327 total cycles.
For more information about each SBS reagent kit, including how to prepare, combine, and split SBS reagents when possible, visit the support page for each kit; also refer to the bulletin Maximum read length for Illumina sequencing platforms for maintaining high data quality.