multiplexed sequencing is simple with Collibri Stranded RNA Libraries

Collibri Stranded RNA libraries can be multiplexed with single-indexed and dual-indexed libraries. 

  • Multiplex sequencing of single- and dual-indexed libraries lowers the cost of sequencing compared to performing two sequencing runs
  • Specify the i5 index sequence in the sample sheet for Collibri Stranded RNA libraries with combinatorial indexing (CD)

Invitrogen Collibri Stranded RNA Library Prep Kits for Illumina Systems provide high-quality, sequencing-ready libraries in 4.5 hrs (mRNA) to 6.5 hrs (whole transcriptome including rRNA depletion) and are suitable for RNA samples of varying quality, including FFPE samples. The Collibri Stranded RNA kits enable construction of cDNA libraries for strand-specific RNA sequencing using either single- or dual-indexed primers on Illumina next-generation sequencing (NGS) systems.

Single-indexed primers are available in the Combinatorial Dual (CD) index version and dual-indexed primers are available in the Unique Dual (UD) index version.

  • Single indexing (CD) is recommended when short run times are required, as only the i7 index needs to be read
  • Dual indexing (UD) is recommended to protect data integrity of samples by minimizing the effects of index-hopping events

Multiplex sequencing of single- and dual-indexed libraries lowers the cost of sequencing

The increased throughput of NGS systems make multiplex sequencing an appealing option to reduce per-sample sequencing costs. It is possible to multiplex libraries with single- and dual-indexes together into a single sequencing run with one modification to the sample sheet. To pool single-indexed libraries prepared with the Collibri Stranded RNA Library Prep Kits (CD) with dual-indexed libraries, you will need to indicate an i5 index in the sample sheet, using the following sequences:

  • TCTTTCCC for MiSeq™, HiSeq™ 2000, HiSeq™ 2500, and NovaSeq™ 6000 systems (Figure 1)
  • AGATCTCG for iSeq™ 100, MiniSeq™, NextSeq™, HiSeq™ 3000, and HiSeq™ 4000 systems

The reads can then be demultiplexed for downstream analysis, reducing the need for two separate sequencing runs.

The i5 index is consistent among Collibri Stranded RNA Libraries with CD indexes.

Application Note: How to sequence single- and dual-indexed libraries together

Single- and dual-indexed libraries may be pooled together to reduce sequencing costs by modifying one column of the sample sheet.

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