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 sequencing instruments.
The choice of the Lexogen UDI set depends on:
The number of samples to be multiplexed
The index sequencing workflow for the instrument or chemistry version with which 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.
Instrument Provider | Workflow Types | ||
|---|---|---|---|
Forward Strand[Illumina Workflow (A)] | Reverse Complement[Illumina Workflow (B)1] | ||
Illumina |
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
| |
Element Biosciences | AVITI AVITI-LT AVITI24 |
| |
Number of Samples | 4 - 96 | Set A1 | Set B13 |
97 - 384 | Set A1 - A4 | Set A1 - A42,4 | |
385 - 768 | 2x (Set A1 - A4) | 2x (Set A1 - A4)2,4 | |
768 - 1152 | 3x (Set A1 - A4) | 3x (Set A1 - A4)2,4 | |
1153 - 1536 | 4x (Set A1 - A4) | 4x (Set A1 - A4)2,4 | |
Table 1 | Lexogen UDI 12 nt Set selection table based on Instrument Workflow 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 instruments that use the Reverse Complement Workflow, the i5 index sequences must be entered in the reverse complement orientation on the sample sheet for demultiplexing (e.g., using 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 If sequencing libraries with Set B1 UDIs on instruments that use the Forward workflow, the reverse complement of the Set B1 i5 sequence (labeled as “i5rc” in the linked index sequence sheet above) must be used.
4 All 12 nucleotides of the i5 index must be read out for error correction. If using sequencing-provider demultiplexing tools (e.g., BaseSpace Sequence Hub, Bcl2fastq, or bases2fastq) the index read should be analyzed as the reverse complement as shown in the example above (2). 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).