Maximum read length for Illumina sequencing platforms


Maximum supported read length

Illumina platforms and sequencing kits allow for a variety of sequencing applications. To ensure the highest level of quality, Illumina supports reads up to a certain length depending on the sequencing platform and SBS kit version; refer to the How many cycles of SBS chemistry are in my kit? support bulletin. It is possible to choose a longer read length during run setup in Local Run Manager (LRM) or the instrument control software. However, when the read length exceeds the supported length Illumina cannot guarantee maintaining high data quality.   

Table 1. Maximum supported read length for sequencing platforms and SBS reagent kits.

Sequencing Platform SBS Kit Version Maximum Read Length
iSeq™ 100 v1 2 x 151bp
v2 2 x 151bp
MiniSeq™ MO* 2 x 151bp
HO* 2 x 151bp
MiSeq™ v2 2 x 251bp
v3 2 x 301bp
NextSeq™ 500/550 MO* 2 x 151bp
HO* 2 x 151bp
NextSeq 1000/2000 P2, P3 2 x 151bp
HiSeq™ 1000/1500/2000/2500 HO* v3 2 x 101bp
HO* v4 2 x 126bp
RR** v4 2 x 251bp
HiSeq 3000/4000 N/A 2 x 151bp
HiSeq X N/A 2 x 151bp
NovaSeq™ 6000 SP 2 x 251bp
S1, S2, S4 2 x 151bp

* MO: Mid-output / HO: High-output
** Rapid Run

Maximum read length for index reads

  • Instrument control software: enter custom Index Read length during run setup for certain instruments, as shown below in Table 2.
  • LRM: enter up to 100 characters for the maximum index cycles (refer to the Local Run Manager 2: How to use a custom library prep kit support bulletin).
  • To ensure sequencing quality of the Index Read, do not exceed the supported read length.

Table 2. Maximum index length for sequencing platforms.

Sequencing Platform Single or Dual Index (Maximum Length)
iSeq 100 Not restricted by software
MiniSeq v1.X 20bp
MiniSeq v2.X 20 bp
MiSeq Not restricted by software
NextSeq 500/550 v2 NCS 20 bp
NextSeq 500/550 v4 NCS 20 bp
NextSeq 1000/2000 v2 NCS Not restricted by software
HiSeq 2500 Not restricted by software
HiSeq 3000/4000 Not restricted by software
HiSeq X 6 bp Single Index / 2 x 8 bp Dual Index
NovaSeq 6000 < v1.6.0 20bp
NovaSeq 6000 ≥ v1.6.0 Not restricted by software