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 P1, 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 v2: 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).
  • LRM v3: enter up to 20 characters or use manual mode if you need between 20 and 100 bp
  • To ensure sequencing quality of the Index Read, do not exceed the supported read length.

Table 2. Maximum individual index length for sequencing platforms.

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

*When setting up runs in Cloud mode, index length is limited to 10 bp.

**For index length between 20-100 bp, use manual mode as LRM v3 is limited to 20 bp