Description

Lexogen 12 nt Unique Dual Index System (UDI) for RNA-Seq

Lexogen’s UDI 12 nt Unique Dual Indexing Sets feature superior error correction for maximal sequencing data output and are introduced at the PCR step of library preparation of following Lexogen kits:

The 12 nt UDIs, provided as Add-on kits for use with QuantSeq and CORALL, are also compatible with other RNA-Seq library prep protocols.

The sets are also available in the QuantSeq-FWD and CORALL with UDI kits or as UDI 12 nt Unique Dual Indixing Sets containing only primer plates without PCR enzymes for use with:

Introduction

A critical consideration for any multiplexed RNA-Seq workflow is to avoid errors in the index read-out, which can result in the mis-assignment of sequencing reads to the wrong samples. While the majority of the raw reads will have the expected index combinations (Fig. 1A), read mis-assignment can occur on all Illumina platforms. This happens due to two main events: Index Hopping and random Index Sequence Errors.

During Index Hopping an index sequence of one library is incorrectly added to another library which may affect 0.1 – 2 % of all reads [1]. Only the use of Unique Dual Indexing (UDI), where each library in a given pool is barcoded with unique i7 and unique i5 index sequences, unambiguously identifies reads with hopped indices. Such reads are removed from downstream analysis and discarded (Fig. 1C).

Read mis-assignment due to random Index Sequence Errors occurs when an error in one index sequence transforms the index into another one that is present within the same multiplexed sample pool. UDIs resolve such mis-assignment and the read is discarded.

More frequently, an Index Sequence Error results in an index sequence that does not match any other index in the pool, and the read is initially classified as undetermined. If the index sequence in question is different enough from the other index sequences in this pool, then error correction can be applied to recover a significant share of these reads (4 – 7 % of the initial reads, Fig. 1B). The performance of this error correction depends predominantly on the quality of the index design, as deficient index design can result in a higher rate of faulty error correction. Due to their unique design the Lexogen UDI 12 nt Unique Dual Indices minimize the impact of Index Sequence Errors and enable maximal data output gain by error correction.

References

[1] Illumina, Effects of Index Misassignment on Multiplexing and Downstream Analysis (2017) 770-2017-004-D.

023_UDI-12nt_Workflow-Index-errors

Figure 1 | The effects of Index Hopping and Index Sequence Errors in a pool of libraries with Unique Dual Indexing. Read mis-assignment caused by Index Hopping can be avoided by using Unique Dual Indexing (UDI). Reads with hopped indices are irreversibly discarded (C). Reads with random Index Sequence Errors resulting in an index not present in the pool are classified undetermined. Accurate error correction can rescue most of these reads making them available for downstream data analysis (B). The percentage values were derived from an RNA-Seq experiment pooling 96 libraries with Lexogen’s 12 nt UDIs and full 12 nucleotide index read-out on an Illumina NextSeq500.

Universal Application

The Lexogen UDI 12 nt Unique Dual Indexing Add-on Kits are compatible with library prep kits for RNA and DNA sequencing from all vendors utilizing TruSeq™ – compatible stubby adapters (where partial Illumina adapters are introduced during the workflow and completed with the index information during the library amplification step).

Superior Error Correction Maximizes Sequencing Yield

The Lexogen UDI 12 nt Unique Dual Indices are 12 nucleotides (nt) long and designed to maximize inter-index distance for different sample numbers and index read-out lengths. In a typical experiment using the full 12 nt index read-out around 9.1 % of the initial raw reads contain a random Index Sequence Error (Fig. 2A). This renders them undetermined, hence removing these reads from downstream analysis.

Lexogen’s advanced index design enables the rescue of 76 % of these undetermined reads (6.9% of the initial reads), even if multiple nucleotides of the index contain errors. The useful output thereby increases to 97.8 % of the initial reads, an unprecedented performance due to the cutting-edge index design (Fig. 2B).

UDI-12nt_PieChart-read-recovery_400px

Figure 2 | Maximizing read output with Lexogen’s 12 nt UDIs and error correction. 96 multiplexed libraries were sequenced on an Illumina NextSeq500 with 12 nt UDI read-out. A) In a standard RNA-Seq experiment a significant number of reads is undetermined (orange) due to random Index Sequence Errors. B) Lexogen’s 12 nt Unique Dual Indices are optimized for maximal error correction with highest accuracy. Lexogen’s Error Correction Tool allows almost 7% of originally undetermined reads to be confidently rescued and correctly assigned to the respective library.

Scalable Index Read-out Length

The design of Lexogen’s 12 nt UDIs enables scalable read-out lengths of 12, 10, and 8 nucleotides. The UDIs therefore support all kinds of requirements for multiplexing, which depend on experiment type, sequencing equipment, desired read depth, and / or the number of pooled libraries. For small sample sizes (e.g., 24 samples) short indices (e.g. 8 or 10 nt) are sufficient to ensure high accuracy and reliable error correction. For more than 96 samples however, 8 nt index read-out does not allow reliable error correction anymore, and 10 or 12 nt read-outs are required.

While needing slightly more sequencing cycles, 12 nt long index sequences also provide the ability to correct not only one but two (or in very small sets, three) Index Sequence Errors. Adjustable index read-out-length allows tuning your indexing needs to the experiment design, without the need to purchase separate indexing sets.

Nested Index Set Design for Highest Accuracy

To provide optimal index subsets for these varying multiplexing needs, Lexogen has designed the 12 nt UDIs in a nested approach: Small subsets benefit from Lexogen’s nested index system by having the largest inter-index distance and highest error correction capacity while larger subsets provide for higher multiplexing needs.

Moreover, all subsets are nucleotide-balanced at each index position for optimized cluster identification in the NGS run. Using a proprietary algorithm, Lexogen has designed more than 9,216 UDIs (24x 384 subsets) with the capacity of correcting at least one error. Such sets with more than 384 UDIs are available upon request and enable extreme levels of multiplexing while still providing excellent error correction.

025_UDI-12nt_Illustration-Nested-Sets_300px

Figure 3 | Distance and error correction in Lexogen’s nested 12 nt UDI sets. Illustration of inter-index distance (D) and number of possible error corrections (ec) in a nested index set with 12 nt read-out. An optimized set of 384 indices contains a subset of 96 indices with larger distances and enhanced error correction. Within these 96 indices, a subset of 24 indices is optimized even further, while a 4 index subset features the highest possible inter-index distance and error correction capacity.

Conclusion

The Lexogen UDI 12 nt Unique Dual Index system adapts to the user’s needs while always providing highest inter-index distance and maximal error correction capacity. Read mis-assignment due to Index Hopping is avoided, and Index Sequence Errors can be corrected with highest accuracy. Thereby, the system provides the optimized indexing solution for current and future barcoding requirements.

Subset 12 nt 10 nt 8 nt
D ec D ec D ec
384 4 1 3 1 2 1*
     └ 96 5 2 4 1 3 1
           └ 24 6 2 5 2 4 1
                 └ 4 7 3 6 2 5 2

Table 1 | Comparison of distance and error correction capacity in Lexogen’s nested 12 nt UDI sets with 8, 10, and 12 nt read-out. Inter-index distance (D) and number of errors that can be corrected (ec) are compared for subsets of 384, 96, 24, and 4 libraries and the three possible read-out lengths. For smaller subsets (up to 96 samples) a read-out of 8 or 10 nt allows correction of one error and thus recovery of additional reads. Larger subsets require a read-out of 10 or 12 nt to benefit from the error correction. The ec values represent the number of all errors (including substitutions, insertions, and deletions) that can be confidently corrected, except for *. In this case error correction can only address substitutions.

FAQs

Lexogen UDI 12 nt Sets contain a unique nested feature enabling adjustable index read-out lengths (the longer the index read-out, the better the error correction). The UDIs are 12 nucleotides long, however, the superior error correction feature can also be used if only 8 or 10 nucleotides of the index are read out, if the i5 index was read out in the correct orientation see Figure 1 and also FAQ 11).
https://www.lexogen.com/wp-content/uploads/2020/11/Lexogen-12-nt-UDI-Sets_Optional-read-out-lengths.jpg
Figure 1 | Optional read-out lengths for Lexogen UDI 12 nt Sets.

If your library yields are extremely low and insufficient for pooling, reamplification can be performed using the Reamplification Add-on Kit for Illumina (080.96). This kit is available only upon request. Please contact Lexogen at support@lexogen.com for more information.

Please note that the PCR Add-on Kit (Cat. No. 020) cannot be used for reamplification of dual-indexed libraries.

Demultiplexing can be carried out by the standard Illumina pipeline. Index sequences (UDI12A_0001-0384 and UDI12B_0001-0096) are available for download at www.lexogen.com/docs/indexing.

Additionally to the standard error-correction included in the Illumina pipeline, Lexogen’s Index Error Correction Tool (available free of charge) can be used in order to rescue even more reads. Please contact support@lexogen.com for more information.

We do not recommend multiplexing Lexogen libraries with libraries from other vendors in the same sequencing lane.

Though this is possible in principle, specific optimization of index combinations, library pooling conditions, and loading amounts may be required, even for advanced users. Sequencing complex pools that include different library types at different lane shares may have unpredictable effects on sequencing run metrics, read quality, read outputs, and / or demultiplexing performance. Lexogen assumes no responsibility for the altered performance of Lexogen libraries sequenced in combination with external library types in the same lane (or run).

Due to size differences, libraries prepared with the Lexogen Small RNA-Seq Library Prep Kit (or any other small RNA library prep kit) should not be sequenced together with QuantSeq, QuantSeq-Flex, SENSE mRNA, or CORALL libraries. Please refer to the sequencing guidelines for each library type (library adapter details, loading amounts to use, and use of custom sequencing primers, etc.), which are provided in our library prep kit User Guides, and online Frequently Asked Questions (FAQs).

Lexogen UDI 12 nt Unique Dual Indexing Add-on Kits are compatible with QuantSeq FWD and REV (Cat. No. 015, 016), QuantSeq-Flex (Cat. No. 033, 034, 035), CORALL (Cat. No. 095, 096) and SENSE mRNA V2 (Cat. No. 001) Library prep kits.

The Lexogen UDI 12 nt Unique Dual Indexing Sets are not compatible with Small RNA library preparation kit (Cat. No. 052).

The Lexogen UDI 12 nt Unique Dual Indexing Add-on Kits are in theory compatible with all non-Lexogen library prep kits utilizing TruSeq™ – compatible stubby adapters (where partial Illumina adapters are introduced during the workflow and completed with the index information during the endpoint PCR step).

The Lexogen i5 6 nt Dual Indexing Add-on Kits (Cat. No. 047) contain the indexed i5 adapters and PCR buffer (Dual PCR Mix) for dual indexing. They can be used to introduce dual indexing for all Lexogen library prep kits that include the Lexogen i7 6 nt Indices (015, 016, 095, 001). The indices of the i7 and i5 6 nt Index Sets are 6 nucleotides long. The Lexogen UDI 12 nt Unique Dual Indexing Plates contain pre-mixed i5 and i7 adapters, each 12 nucleotides long. The respective Add-on Kits additionally include the PCR enzyme (E) and PCR buffer (Dual PCR Mix) required for dual indexing.

When using the 12 nt UDI Add-on Kits (Cat. No. 107-111, 120) for introduction of Unique Dual Indexing to libraries prepared with Lexogen kits that include the Lexogen i7 6 nt Indices (015, 016, 095, 001), the PCR Mix (yellow) from the library prep kits has to be replaced by the Dual PCR Mix (purple) from the UDI 12 nt Add-on kits and the 12 nt UDIs are used instead of the i7 6 nt indices.

For convenience, the QuantSeq FWD (113-115 and 129-131), CORALL Total RNA-Seq (117-119 and 132-134), and CORALL mRNA-Seq (Cat. No. 158-163) kits include the 12 nt UDI Sets instead of the i7 6 nt Index Sets.

Unsure which 12 nt UDI Set to use? Please consult the or contact support@lexogen.com.

  1. 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.
  2. 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.

Number of Samples Illumina Instrument
Forward Strand Workflow (A) Reverse Complement Workflow (B)
HiSeq 2000/2500
HiSeq 3000/4000
HiSeq 2000/2500
MiSeq
NovaSeq 6000 (v1.0 reagent kits)
iSeq 100
MiniSeq
NextSeq 500 – 2000
HiSeq 3000/4000
NovaSeq 6000 (v1.5 reagent kits)
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

.
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. 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.
2 All 12 nucleotides of the i5 index must be read out for error correction using Lexogen’s Error Correction Tool and the index read should be analyzed as the reverse complement, in this case CCCAGTTACTAA from the example above (i512A_0002).
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-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 3’ 2
Cat. No. 143
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-Rev3
Cat. No. 016
Cat. No. 107: Set A1
Cat. No. 108: Set A2
Cat. No. 109: Set A3
Cat. No. 110: Set A4
Cat. No. 120: Set A1-A4
Cat. No. 111: Set B1
QuantSeq-Flex3
Cat. No. 033-035
Cat. No. 107: Set A1
Cat. No. 108: Set A2
Cat. No. 109: Set A3
Cat. No. 110: Set A4
Cat. No. 120: Set A1-A4
Cat. No. 111: Set B1
Other Vendor Libraries4 Cat. No. 107: Set A1
Cat. No. 108: Set A2
Cat. No. 109: Set A3
Cat. No. 110: Set A4
Cat. No. 120: Set A1-A4
Cat. No. 111: Set B1
QuantSeq-FWD
Cat. No. 015
Cat. No. 113-115, 129-131
CORALL
Cat. 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 don’t contain 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.

The NovaSeq Xp workflow enables individual lane loading on the same flow cell. Hence the multiplexing capacity can be expanded 2 times using the NovaSeq XP 2-Lane Kit or 4 times with the NovaSeq XP 4-Lane Kit. Hence, it is possible load up to 4 physically separated 384-plex UDI-indexed libraries per flow cell. For more information about the Xp Workflow, please refer to the NovaSeq™ Xp workflow Technical Note, The NovaSeq™ Xp workflow explanatory video, or contact Illumina Support.

Yes. The NovaSeq 6000 v1.5 Reagent Kits include additional SBS chemistry cycles, which are increased from 30 to 38 compared to the previous v1.0 Reagent Kits. Thus, expanding the maximum number of supported cycles per kit to 538, 338, 238, and 138 for 500, 300, 200, and 100-cycle kits, respectively. Moreover, in the new v1.5 Kits, the Reverse Complement Workflow (B) replaces the Forward Strand Workflow (A) utilized by the v1.0 Kits. This change further impacts the number of effective SBS chemistry cycles because the Reverse Complement Workflow does not require additional seven cycles of reagents for the chemistry-only cycles at the beginning of the second Index Read. Therefore, with additional 38 effective SBS chemistry cycles, as opposed to only 23 with v1.0 Kits, one can comfortably read out 12 + 12 nt from the Lexogen UDIs. For more information please refer to the How many cycles of SBS chemistry are in my kit? Illumina bulletin or contact Illumina Support.

Lexogen offers two tools (available free of charge) that can be used for highest accuracy in error correction. The Index Error Correction Tool is the method of choice for Single-Read (SR) data,  i1 Demultiplexing Tool (iDEMUX) is recommended for Paired-End (PE) data and required for QuantSeq-Pool.

Suitable Tool Sequencing Format Indexing Type Index read-out length Compatible Library Prep Kits
Index Error Correction Tool Single-Read (SR) Dual-Indexing (i7 and i5) 8 nt1
10 nt1
12 nt
QuantSeq FWD
QuantSeq REV (SR)
QuantSeq-Flex
CORALL (SR)
LUTHOR 3’ (SR)
Other vendors (SR)
i1 Demultiplexing Tool (iDEMUX)2 Paired-End (PE) Dual Indexing (i7 and i5)
Triple-Indexing (i7, i5 and i1)3
8 nt
10 nt
12 nt
QuantSeq-Pool
QuantSeq REV (PE)
CORALL (PE)
Other vendors (PE)

.
Table 3 | Error correction tool selection criteria.

1 Error correction for index read out lengths <12 nt can only be performed with this tool if the i5 index is read out in the correct orientation e.g., when libraries indexed with UDI Sets A1-A4 are sequenced on Forward Strand Workflow Illumina instruments, or when libraries indexed with UDI Set B1 sequenced on Reverse Complement Workflow Illumina instruments. See Table 1. from FAQ 8. for a list of Illumina instruments grouped by dual-indexing workflow.
2 The i1 Demultiplexing Tool allows demultiplexing and error correction in one go.
3 The i1 index, and thus triple indexing, is a feature that is currently only present in QuantSeq-Pool libraries.

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