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.
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) | |
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) 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 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 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 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 How can I multiplex more than 384 samples on an Illumina NovaSeq instrument if you only offer 384 different UDIs?).
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 use.
Table 2: Lexogen UDI 12 nt Unique Dual Indexing product compatibility with Library Prep Kits.
Library Prep Type |
Base Kit(s)with 6 nt indices or no indices included. | UDI Products | ||
UDI Primer Plate Only | 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 | ||
LUTHOR2 | Cat. No. 143 | AND 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 | ||
QuantSeq FWD3 | Cat. No. 015 |
| AND one of: 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 | OR one of the following UDI Kit Bundles: Cat. No. 191 - 196 |
QuantSeq-REV3
| Cat. No. 016 | AND one of: 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 | ||
QuantSeq-Flex3 | Cat. No. 166 AND/OR Cat. No. 028 |
| AND one of the following QuantSeq FWD UDI Kit Bundles Cat. No. 191 - 196 | |
CORALL | Cat. No. 095 (stand alone kit) OR Cat. No 144 - 146 (RiboCop bundles) |
| AND one of: 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 | OR one of the following UDI Kit Bundles: Cat. No. 171 – 176 - stand alone kits Cat. No. 177 - 182 - with Poly(A) RNA Enrichment Cat. No. 183 - 186 - with RiboCop rRNA Depletion |
Other Vendor Libraries4 | NA |
| OPTIONALLY one of: 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 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).
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.