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?|
|HiSeq - TruSeq SBS v3||50||58||7 cycles required for paired-end flow cells|
|HiSeq - HiSeq SBS v4||50||79|
|HiSeq - Rapid SBS v2||50||79|
|MiSeq v2 (including Micro and Nano kits)||50||79||Yes - 7 cycles|
|NovaSeq 6000 SP||100||130|
|NovaSeq 6000 S1 and S2||100||130|
|NovaSeq 6000 S4||200||230|
|HiSeq 3000/4000 SBS||50||79||7 cycles required for single-read flow cells|
|iSeq 100 i1||300||322||No|
|MiniSeq High Output or Mid Output||75||92|
|NextSeq 500/550 High Output or Mid Output||75||92|
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.