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 - A4^1,2
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

¹When 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 sequencesmust 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. here.

²All 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?).

⁶These 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)

NOTE: The UDI Add-on Kits can be used in combination with Base Kits that contain no indices or that contain 6 nt i7 indices. When using the Base Kits indicated below, one of the UDI Primer Plates or Add-on Kits will be needed in addition. However, if UDIs should be included in libraries, we recommend purchasing the Kit Bundles for ease of useThe 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)

with 6 nt indices or no indices included.

UDI Products

UDI Primer Plate Only³

UDI Add-on Kits

(Primer Plate, Buffer, and Enzyme)

UDI Kit Bundles

(Library Prep Kit + UDI Primer Plate, Buffer, and Enzyme)

QuantSeq-Pool¹

Cat. No. 139

OPTIONALLY one of:

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

²

HD

Cat. No.

143AND one of:

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

QuantSeq FWD³

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.

015 AND one of:

156: Set A1 – A4

Cat. No. 105: Set B1

Other Vendor Libraries²

N/A

Cat. No.

107

198: Set A1

Cat. No.

108

199: Set A2

Cat. No.

109

200: Set A3

Cat. No.

110

201: Set A4

Cat. No.

120

203: Set A1 - A4

Cat. No.

111

202: Set B1

OR one of the following UDI Kit Bundles:

Cat. No. 113 – 115, 129 – 131

QuantSeq-REV³

Cat. No. 016

AND one of:

Cat. No. 107: Set A1

Cat. No. 108:

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.

³Each 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.

109:

195.96	Set A3
Cat. No. 196.

110:

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.

120:

222.4x96

Set A1

– A4

- A4

Cat. No. 223.24

Cat. No.

111:

223.96	Set B1
QuantSeq

-Flex³

3’ mRNA-Seq V2 Library Prep Kit REV with UDI

Cat. No

. 166 AND/OR

. 225.24

Cat. No.

028AND one of the following QuantSeq FWD UDI Kit Bundles

225.96	Set B1
Whole Transcriptome Library Prep Kits	CORALL RNA-Seq V2 Library Prep Kit with UDI	Cat. No.

113 – 115, 129 – 131

CORALL V2

NA

171.24

Cat. No. 171.96

Set A1

Cat. No.

171 – 176 - stand alone kits

172.96	Set A2
Cat. No.

177 - 182 - with Poly(A) RNA Enrichment

173.96	Set A3
Cat. No

. 183 - 186 - with RiboCop rRNA DepletionCORALL V1

. 174.96

Set A4

Cat. No. 175.24

Cat. No

. 095 (stand alone kit) OR

. 175.96	Set B1
Cat. No

144 - 146 (RiboCop bundles)AND one of:

. 176.384	Set A1 - A4
CORALL mRNA-Seq V2 Library Prep Kit with UDI	Cat. No. 177.

107:

96	Set A1
Cat. No.

108:

178.96	Set A2
Cat. No. 179.

109:

96	Set A3
Cat. No.

110:

180.96	Set A4
Cat. No. 181.96	Set B1
Cat. No. 182.

120:

384

Set A1 - A4

RiboCop (HMR) and CORALL Total RNA-Seq V2 Library Prep Kit with UDI

Cat. No. 183.24

Cat. No. 183.

111: Set B1OR one of the following UDI Kit Bundles:

96	Set A1
Cat. No.

117 - 119, 132 - 134 - stand alone kits

184.24

Cat. No

. 158 - 163 - with Poly(A) RNA Enrichment

Other Vendor Libraries⁴

NA

OPTIONALLY one of:

. 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

. 107: Set A1

. 186.24

Cat. No.

108: Set A2

186.96	Set B1
CORALL FFPE Whole Transcriptome RNA-Seq Library Prep with UDI	Cat. No.

109: Set A3

219.24

Cat. No. 219.

110:

96

Set

A4

A1

Cat. No.

120:

219.4x96

Set A1 - A4

Cat. No. 220.24

Cat. No. 220.

111:

96

Set B1

¹ QuantSeq-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 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 CORALL V1; QuantSeq FWD, REV, and -Flex kits have to be replaced by the amplification reagents provided in the UDI Add-on kits (as instructed in the UDI Add-on Kit Instruction Manual).

...

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).

Versions Compared

Old Version 14

New Version Current

Key

Choosing the correct UDI Set

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)

Unique Dual Indexing Product Offerings & Compatibility

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

UDI Kit Bundles

Table 3: Lexogen Library Prep Kits including 12 nt UDIs (UDI Kit Bundles)

Product Name

Catalog Number¹

UDI Set

Expression Profiling Library Prep Kits

Whole Transcriptome Library Prep Kits

Single-cell and Low-input RNA-Seq Library Prep Kits

Page Comparison

Versions Compared

Old Version 14

New Version Current

Key

Choosing the correct UDI Set

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)

Unique Dual Indexing Product Offerings & Compatibility

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

UDI Kit Bundles

Table 3: Lexogen Library Prep Kits including 12 nt UDIs (UDI Kit Bundles)

Product Name

Catalog Number1

UDI Set

Expression Profiling Library Prep Kits

Whole Transcriptome Library Prep Kits

Single-cell and Low-input RNA-Seq Library Prep Kits

UDI Primer Plate Only³

Catalog Number¹