Description

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

Lexogen’s UDI 12 nt Unique Dual Indexing Sets are provided as Add-on Kits and are introduced at the PCR step of library preparation of QuantSeq 3’ mRNA-Seq FWD and REV, QuantSeq-Flex, CORALL Total RNA-Seq, and SENSE mRNA-Seq V2 for Illumina. The Add-on kits are also compatible with other RNA-Seq library prep protocols. The 12 nt Unique Dual Indexing Sets are also available in the QuantSeq-FWD and CORALL with UDI kits.

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.

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 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 “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 contain the indexed i5 adapters only. 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 Kits contain pre-mixed i5 and i7 adapters, each 12 nucleotides long.

When using the UDI 12 nt Sets 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 (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 and CORALL kits are available as versions containing the UDI 12 nt Sets instead of the i7 6 nt Index Sets (113-115 and 117-119, respectively).

Downloads

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

pdf Instruction Manual for Lexogen 12 nt Unique Dual Indexing Add-on Kit – release 27.12.2019
pdf User Guide for QuantSeq 3‘ mRNA-Seq Library Prep Kit FWD with Unique Dual Indices – release 27.12.2019
pdf User Guide for CORALL Total RNA-Seq Library Prep Kit with Unique Dual Indices – release 27.12.2019

pdf Product Flyer

pdf Lexogen UDI 12 nt Unique Dual Index Sequences – release 11.12.2019

Index Error Correction Tool

Material Safety Datasheets

pdf MSDS information for UDI 12nt Unique Dual Indexing Add-On Kits – release 17.12.2019

If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.

Buy from our Webstore

Need a web quote?

You can generate a web quote by Register or Login to your account. In the account settings please fill in your billing and shipping address. Add products to your cart, view cart and click the “Generate Quote” button. A quote in PDF format will be generated and ready to download. You can use this PDF document to place an order by sending it directly to sales@lexogen.com.

Web quoting is not available for countries served by our distributors. Please contact your local distributor for a quote.