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Can I use the SI Primer together with Dual Index Plate TT when preparing Single Cell 3' Gene Expression libraries?

Question: Can I use the SI Primer together with Dual Index Plate TT when preparing Single Cell 3' Gene Expression libraries?

Answer: The SI Primer should not be used together with Dual Index Plate TT. This primer should only be used together with Single Index Plate T. 

Different primers are required in the Sample Index PCR step for generating single index and dual index Single Cell 3' Gene Expression libraries. Please follow the appropriate User Guide based on your library type. 

Single index libraries can be generated using the following primers:

  • SI Primer (PN-2000095)
    • This is a forward primer containing P5 and Read1 sequences
  • Single Index Plate T Set A (PN-1000213/2000240)
    • Each well of this index plate contains reverse primers with P7, Read2, and i7 sample index sequences

Dual index libraries can be generated using the following primers:

  • Dual Index Plate TT Set A (PN-1000215/3000431)
    • Each well of this index plate contains a mixture of forward and reverse primers
    • The forward primer contain P5, Read1 and i5 index sequences
    • The reverse primer contains P7, Read2 and i7 index sequences 

SIPrimers.png

 

If the SI Primer is mistakenly used together with the Dual Index Plate TT Set A, you will end up generating a mixture of dual index and single index library fragments, as show here:

 

incorrect_primers_SIPrimer.png

If the fragments that contain a single index are sequenced using dual index parameters, the i7 index read will contain the expected i7 sequence, but the i5 index read will contain adapter sequences (given that there is no i5 index). These adapter sequences will be as follows:

  • AGATCTCGGT if using Illumina's Reverse Complement Sequencing Workflow
  • TCTTTCCCTA if using Illumina's Forward Sequencing Workflow

To rescue sequencing data from such an experiment, you can perform demultiplexing based on the i7 index only. 

If the libraries have not yet been sequenced, we recommend re-preparing the libraries from left over cDNA.

Also see: What are the protocol differences between Single Cell 3' v3.1 Single Index and 3' v3.1 Dual Index?

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