- 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.
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.
[Image2]
Table 1 | Lexogen UDI 12 nt Set selection table based on Illumina Instrument and number of samples.
1 When sequencing libraries indexed with UDI Sets A1-A4 on Illumina instruments that use the Reverse Complement Workflow for dual-indexing, 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 at www.lexogen.com/docs/indexing.
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 FAQ 9).
4 Loading 3 individual lanes on a NovaSeq Xp 4-Lane Kit using the Xp Workflow (See FAQ 9).
5 Loading 4 individual lanes on a NovaSeq Xp 4-Lane Kit using the Xp Workflow (See FAQ 9).
There are three Lexogen UDI 12 nt Unique Dual Indexing product categories: Primer Plate Only (amplification reagents not included), Add-on Kit (primer plate + amplification reagents), and bundles (library prep kit + primer plate + amplification reagents).
Library Prep Kit |
UDI product |
|
|
|
Primer Plate Only |
Add-on Kit(Primer Plate, Buffer, Enzyme) |
Bundle(Library Prep Kit + Primer Plate, Buffer and Enzyme) |
QuantSeq-Pool1Cat. No. 139 |
Cat. No. 101: Set A1Cat. No. 102: Set A2Cat. No. 103: Set A3Cat. No. 104: Set A4Cat. No. 156: Set A1-A4Cat. No. 105: Set B1 |
|
|
LUTHOR 3' 2Cat. No. 143 |
Cat. No. 101: Set A1Cat. No. 102: Set A2Cat. No. 103: Set A3Cat. No. 104: Set A4Cat. No. 156: Set A1-A4Cat. No. 105: Set B1 |
|
|
QuantSeq-Rev3Cat. No. 016 |
|
Cat. No. 107: Set A1Cat. No. 108: Set A2Cat. No. 109: Set A3Cat. No. 110: Set A4Cat. No. 120: Set A1-A4Cat. No. 111: Set B1 |
|
QuantSeq-Flex3Cat. No. 033-035 |
|
Cat. No. 107: Set A1 Cat. No. 108: Set A2Cat. No. 109: Set A3Cat. No. 110: Set A4Cat. No. 120: Set A1-A4Cat. No. 111: Set B1 |
|
Other Vendor Libraries4 |
|
Cat. No. 107: Set A1Cat. No. 108: Set A2Cat. No. 109: Set A3Cat. No. 110: Set A4Cat. No. 120: Set A1-A4Cat. No. 111: Set B1 |
|
QuantSeq-FWDCat. No. 015 |
|
|
Cat. No. 113-115, 129-131 |
CORALLCat. No. 095 |
|
|
Cat. No. 117-119, 132-134 |
. Table 2 | Lexogen UDI 12 nt Unique Dual Indexing product compatibility with Library Prep Kits.
1 QuantSeq-Pool kits contain 96 inline barcodes (i1) for individual sample barcoding before pooling. For analysis of more than 96 samples additional indexing with e.g. UDIs is required.
2 LUTHOR kits do not include barcodes. Additional purchase is required for functionality of the library prep.
3 The amplification reagents which are provided in the QuantSeq-Rev and QuantSeq-Flex kits have to be replace by the amplification reagents provided in the UDI Add-on kits (as instructed in the UDI Add-on Kit Instruction Manual).
4 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.