How do I choose the right Lexogen UDI 12 nt Unique Dual Indexing product for my libraries?
Choose one or more compatible UDI Sets depending on the number of samples to be multiplexed and the Illumina instrument (using Forward Strand or Reverse Complement Workflow for dual-indexing) where the libraries will be sequenced. See Table 1 for details.
Choose the right product (Primer Plate, Add-on Kit or Bundle) based on the Library Prep Kit (Lexogen or other vendors). See Table 2 for a list of Library Prep Kits and the appropriate UDI product (with Catalog Number) to be indexed with, and see Table 3 for a list of our Library Prep Kits bundled with UDIs.
Choosing the correct UDI Set
The Lexogen UDI 12 nt Unique Dual Indexing Sets are available in different sets (A1-A4 and B1) for optimal compatibility with different Illumina sequencing instruments.
Table 1: Lexogen UDI 12 nt Set selection table based on Illumina Instrument and number of samples.
Number of Samples | Illumina Instrument | |
|---|---|---|
Forward Strand Workflow (A) | Reverse Complement Workflow (B)6 | |
HiSeq 2000/2500 HiSeq 3000/4000 (Single-Read Flow Cell) MiSeq MiniSeq (Rapid Reagent Kits) NovaSeq 6000 (v1.0 Reagent Kits) | iSeq 100 HiSeq 3000/4000 (Paired-End Flow Cell) NextSeq 500 - 2000 MiniSeq (Standard Reagent Kit) NovaSeq 6000 (v1.5 Reagent Kits) NovaSeq X / X Plus HiSeq X | |
4 - 96 | Set A1 | Set B1 |
97 - 384 | Set A1 - A4 | Set A1 - A41,2 |
385 - 768 | 2x (Set A1 - A4)3 | 2x (Set A1 - A4)1,2,3 |
768 - 1152 | 3x (Set A1 - A4)4 | 3x (Set A1 - A4)1,2,4 |
1153 - 1536 | 4x (Set A1 - A4)5 | 4x (Set A1 - A4)1,2,5 |
1 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.
2 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, in this case CCCAGTTACTAA from the example above (i512A_0002). Alternatively, if using Lexogen’s Error Correction Tool idemuxCPP demultiplexing / error correction tool, do not enter the reverse complement sequences (i5rc) into the sample sheet, instead to use the --i5-rc parameter. See idemuxCPP documentation at https://github.com/Lexogen-Tools/idemuxcpp .
3 Loading 2 individual lanes on a NovaSeq Xp 2-Lane or 4-Lane Kit using the Xp Workflow (See Working Copy How can I multiplex more than 384 samples on an Illumina NovaSeq instrument if you only offer 384 different UDIs?).
4 Loading 3 individual lanes on a NovaSeq Xp 4-Lane Kit using the Xp Workflow (See Working Copy How can I multiplex more than 384 samples on an Illumina NovaSeq instrument if you only offer 384 different UDIs?).
5 Loading 4 individual lanes on a NovaSeq Xp 4-Lane Kit using the Xp Workflow (See Working Copy How can I multiplex more than 384 samples on an Illumina NovaSeq instrument if you only offer 384 different UDIs?).
6 These instruments use the reverse complement workflow, but the i5rc sequence is required to be entered if the analysis options available on the instrument (BaseSpace or Onboard instrument Dragen analysis) are used to generate a sample sheet. You will always be able to re-demultiplex with an updated sample sheet if modifications are required. Please review this article for a table of which instruments and softwares require which i5 index orientation or contact Illumina for additional support.
Unique Dual Indexing Product Offerings & Compatibility
There are three Lexogen UDI 12 nt Unique Dual Indexing product categories:
UDI Primer Plate Only (amplification reagents not included)
UDI Add-on Kits (primer plate + amplification reagents)
UDI Kit Bundles (library prep kit + primer plate + amplification reagents)
The following table provides an overview on compatibility of library prep kits, which contain no indices (i.e., Base Kits), with our Unique Dual Indexing products.
Table 2: Lexogen UDI 12 nt Unique Dual Indexing product compatibility with Library Prep Kits without bundled indices (Base Kits).
Library Prep Type |
Base Kit(s) | UDI Products | |
UDI Primer Plate Only3 | UDI Add-on Kits(Primer Plate, Buffer, and Enzyme) | ||
QuantSeq-Pool1 | Cat. No. 139 | Cat. No. 101: Set A1 Cat. No. 102: Set A2 Cat. No. 103: Set A3 Cat. No. 104: Set A4 Cat. No. 156: Set A1 - A4 Cat. No. 105: Set B1 |
|
LUTHOR HD | Cat. No. 204
| Cat. No. 101: Set A1 Cat. No. 102: Set A2 Cat. No. 103: Set A3 Cat. No. 104: Set A4 Cat. No. 156: Set A1 – A4 Cat. No. 105: Set B1 |
|
LUTHOR HD Pool1 | Cat. No. 205 | Cat. No. 101: Set A1 Cat. No. 102: Set A2 Cat. No. 103: Set A3 Cat. No. 104: Set A4 Cat. No. 156: Set A1 – A4 Cat. No. 105: Set B1 |
|
Other Vendor Libraries2 | N/A |
| Cat. No. 198: Set A1 Cat. No. 199: Set A2 Cat. No. 200: Set A3 Cat. No. 201: Set A4 Cat. No. 203: Set A1 - A4 Cat. No. 202: Set B1 |
1 QuantSeq-Pool and LUTHOR HD Pool kits contain 96 in-line indices (i1) for individual sample barcoding before pooling. For analysis of more than 96 samples, additional indexing with e.g., UDIs is required.
2 Libraries (RNA-Seq and DNA-Seq) from other vendors must contain TruSeq™ – compatible stubby adapters (where partial Illumina adapters are introduced during the workflow and completed with the index information during the endpoint PCR step). When unsure about compatibility please contact support@lexogen.com.
3 Each individual Set contains 96 UDIs.
Table 3: Lexogen Library Prep Kits including 12 nt UDIs (UDI Kit Bundles)
| Product Name | Catalog Number1 | UDI Set |
|---|---|---|---|
Expression Profiling Library Prep Kits | QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI | Cat. No. 191.24 Cat. No. 191.96 | Set A1 |
Cat. No. 192.24 Cat. No. 192.96 | Set B1 | ||
Cat. No. 193.384 | Set A1 - A4 | ||
Cat. No. 194.96 | Set A2 | ||
Cat. No. 195.96 | Set A3 | ||
Cat. No. 196.96 | Set A4 | ||
QuantSeq FFPE 3’ mRNA-Seq Library Prep Kit FWD with UDI | Cat. No. 222.24 Cat. No. 222.96 | Set A1 | |
Cat. No. 222.4x96 | Set A1 - A4 | ||
Cat. No. 223.24 Cat. No. 223.96 | Set B1 | ||
QuantSeq 3’ mRNA-Seq V2 Library Prep Kit REV with UDI | Cat. No. 225.24 Cat. No. 225.96 | Set B1 | |
Whole Transcriptome Library Prep Kits | CORALL RNA-Seq V2 Library Prep Kit with UDI | Cat. No. 171.24 Cat. No. 171.96 | Set A1 |
Cat. No. 172.96 | Set A2 | ||
Cat. No. 173.96 | Set A3 | ||
Cat. No. 174.96 | Set A4 | ||
Cat. No. 175.24 Cat. No. 175.96 | Set B1 | ||
Cat. No. 176.384 | Set A1 - A4 | ||
CORALL mRNA-Seq V2 Library Prep Kit with UDI | Cat. No. 177.96 | Set A1 | |
Cat. No. 178.96 | Set A2 | ||
Cat. No. 179.96 | Set A3 | ||
Cat. No. 180.96 | Set A4 | ||
Cat. No. 181.96 | Set B1 | ||
Cat. No. 182.384 | Set A1 - A4 | ||
RiboCop (HMR) and CORALL Total RNA-Seq V2 Library Prep Kit with UDI | Cat. No. 183.24 Cat. No. 183.96 | Set A1 | |
Cat. No. 184.24 Cat. No. 184.96 | Set B1 | ||
RiboCop (HMR+Globin) and CORALL Total RNA-Seq V2 Library Prep Kit with UDI | Cat. No. 185.24 Cat. No. 185.96 | Set A1 | |
Cat. No. 186.24 Cat. No. 186.96 | Set B1 | ||
CORALL FFPE Whole Transcriptome RNA-Seq Library Prep with UDI | Cat. No. 219.24 Cat. No. 219.96 | Set A1 | |
Cat. No. 219.4x96 | Set A1 - A4 | ||
Cat. No. 220.24 Cat. No. 220.96 | Set B1 | ||
RiboCop and CORALL FFPE Whole Transcriptome RNA-Seq Library Prep Kit with UDI | Cat. No. 233.24 Cat. No. 233.96 | Set A1 | |
Cat. No. 234.24 Cat. No. 234.96 | Set B1 | ||
Single-cell and Low-input RNA-Seq Library Prep Kits | LUTHOR High-Definition Single-Cell 3' mRNA-Seq Kit with UDI | Cat. No. 221.24 Cat. No. 221.96 | Set B1 |
1Catalog numbers indicate number of reactions in each kit e.g., Cat. No. xxx.24 contains 24 reactions (=24 UDIs).