How to choose the right Lexogen UDI 12 nt Unique Dual Indexing set?
The Lexogen 12 nt Unique Dual Indexing Sets are available in different sets (A1 - A4 and B1) for optimal compatibility with different Illumina sequencing instruments.
The choice of the Lexogen UDI set depends on:
The number of samples to be multiplexed
The Illumina instrument where the libraries will be sequenced (i.e., using Forward Strand or Reverse Complement Workflow for dual-indexing - See Table 1 for details).
NOTE: For detailed information on how indices are sequenced on different Illumina platforms, please see the following overview guide from Illumina.
Number of Samples | Illumina Instrument | |
|---|---|---|
Forward Strand Workflow (A) | Reverse Complement Workflow (B)1 | |
MiSeq i 100/ i100 Plus (Index-first sequencing mode) MiniSeq (Rapid Reagent Kits) | iSeq 100 MiSeq i 100/ i100 Plus (Read-first sequencing mode) NextSeq 500 - 2000 MiniSeq (Standard Reagent Kit) NovaSeq 6000 (v1.5 Reagent Kits) NovaSeq X / X Plus | |
4 - 96 | Set A1 | Set B1 |
97 - 384 | Set A1 - A4 | Set A1 - A42,3 |
385 - 768 | 2x (Set A1 - A4) | 2x (Set A1 - A4)2,3 |
768 - 1152 | 3x (Set A1 - A4) | 3x (Set A1 - A4)2,3 |
1153 - 1536 | 4x (Set A1 - A4) | 4x (Set A1 - A4)2,3 |
Table 1 | Lexogen UDI 12 nt Set selection table based on Illumina Instrument and number of samples.
IMPORTANT!
1 These instruments use the reverse complement workflow. For demultiplexing, please review this article to determine which i5 index orientation (forward or reverse) must be used for demultiplexing based on the analysis options available on the instrument (i.e., the demultiplexing software).
2 When sequencing libraries indexed with UDI Sets A1-A4 on Illumina instruments that use the Reverse Complement Workflow (and vice versa for Set B1 UDIs used for Forward Strand Workflow), the i5 index sequences must be entered in the reverse complement orientation on the sample sheet for demultiplexing with Illumina demultiplexing tools (e.g., BaseSpace Sequence Hub or Bcl2fastq). EXAMPLE: i512A_0002 should be entered as TTAGTAACTGGG instead of CCCAGTTACTAA in the sample sheet for demultiplexing. The reverse complement sequences (i5rc) are provided the index sequence sheet available for download here.
3 All 12 nucleotides of the i5 index must be read out for error correction. If using Illumina demultiplexing tools (e.g., BaseSpace Sequence Hub or Bcl2fastq) the index read should be analyzed as the reverse complement as shown in the example above. Alternatively, if using Lexogen’s Demultiplexing and Error Correction Tool enter the forward sequences into the sample sheet and use the following --i5-rc parameter (https://github.com/Lexogen-Tools/idemuxcpp).