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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).

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