Description

QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

The QuantSeq REV Kit is a library preparation protocol designed to generate Illumina compatible libraries of sequences at the 3’ end of the polyadenylated RNA.

With QuantSeq Reverse (REV) it is possible to exactly pinpoint the 3’ end in Read 1, hence to study the 3’ UTR and alternative polyadenylation. With the help of the Custom Sequencing Primer (CSP Version 2, included in the kit) the first nucleotide of your NGS read corresponds the very last nucleotide of the mRNA. The reads generated during Read 1 reflect the cDNA sequence.

Analysis of Low Input and Low Quality Samples

The required input amount of total RNA is as low as 10 ng. QuantSeq is suitable to reproducibly generate libraries from low quality RNA, including FFPE samples.

Rapid Turnaround

QuantSeq’s simple workflow allows generating ready-to sequence NGS libraries within only 4.5 hours, including less than 2 hours hands-on time.

Deplete globin mRNAs during QuantSeq Library Prep

New! Globin Block Modules for QuantSeq enable the generation of globin-depleted, ready-to-sequence 3’ mRNA-Seq libraries from as little as 50 ng of total RNA from whole blood. No additional protocol steps are required. The RS-Globin Block solution from the module simply replaces the standard RNA Removal Solution from the QuantSeq Kit. Using Globin Block for QuantSeq, reads mapping to globin mRNAs can be reduced from 50 – 80 %, to as low as 5% (Fig. 1) and gene detection is dramatically increased (Fig.2).
* For Figures: Input RNA (50 ng, or 250 ng) was extracted with: 1 SPLIT RNA Extraction Kit, 2 other kit including red blood cell lysis, or 3 other kit without red blood cell lysis.
Fig.1-Globin-Block_1000px

Figure 1 | Percentage of sequencing reads mapping to globin mRNAs from human (Hs) and pig (Ss) blood QuantSeq libraries.*

Fig.2-Globin-Block_1000px

Figure 2 | Increased gene detection in human blood QuantSeq libraries using Globin Block. CPM = Counts Per Million.*

High Strand-Specificity

QuantSeq maintains exceptional strand-specificity of >99.9 % and allows to map reads to their corresponding strand on the genome, enabling the discovery and quantification of antisense transcripts and overlapping genes.

Simple Bioinformatics Analysis

Read mapping is simplified by skipping the junction detection. Reads are generated at the transcripts’ most 3′ end where nearly no junctions are located. Data processing can hence be accelerated by using e.g., Bowtie2 instead of TopHat2.

The QuantSeq data analysis pipeline has been integrated on the Bluebee® genomics analysis platform and is accessible for user even without bioinformatics background. Access to the pipeline is free of charge for any QuantSeq 3’ mRNA-Seq for Illumina customers. Learn more and get started at https://www.bluebee.com/lexogen/

Mapping of Transcript End Sites

QuantSeq allows to exactly pinpoint the 3’ end of poly(A) RNA and therefore obtain accurate information about the 3’ UTR.

Direct Counting for Gene Expression Quantification

Just one fragment per transcript is produced; therefore no length normalization is required. This allows more accurate determination of gene expression values and renders QuantSeq the best alternative to microarrays and conventional RNA-Seq in gene expression and eQTL studies.

Cost Saving Multiplexing

QuantSeq libraries are intended for a high degree of multiplexing. With the up to 96 i7 indices (7001-7096) included in the kit and the additionally available 96 i5 indices (Lexogen i5 6 nt Unique Dual Indexing Add-on Kit (5001-5096) (Cat. No. 047)), up to 9,126 samples can be multiplexed and sequenced per lane on an Illumina flow cell.
This high level of multiplexing allows saving costs as the length restriction in QuantSeq saves sequencing space. QuantSeq is also designed to yield insert sizes for short sequencing reads (SR50, SR100).

For the detailed information about barcodes and instructions how to use them please consult Appendix K: Multiplexing, QuantSeq for Illumina User Guide (p. 35); Appendix A: Multiplexing, Lexogen i5 6 nt Dual Indexing Add-on Kit Instruction Manual (p. 7).

Workflow

QuantSeq has a short and simple workflow and can be completed within 4.5 hours. The required hands-on time is less than 2 hours. The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.

Reverse Transcription
Step 1:
The kit uses total RNA as input, hence no prior poly(A) enrichment
or rRNA depletion is needed.
01quantseq_workflowtime_quantseq01
Reverse Transcription
Step 1:
Library generation starts with oligodT priming containing the
Illumina-specific Read 1 linker sequence.
02quantseq_workflow_revtime_quantseq02
Removal of RNA
Step 2:
After first strand synthesis the RNA is removed.
03quantseq_workflow_revtime_quantseq03
Second-Strand Synthesis
Step 3:
Second strand synthesis is initiated by random priming and a DNA
polymerase. The random primer contains the Illumina-specific Read 2
linker sequence.
04quantseq_workflow_revtime_quantseq04
Second-Strand Synthesis
Step 3:
No purification is required between first and second strand synthesis.
Second strand synthesis is followed by a magnetic bead-based
purification step rendering the protocol compatible with automation.
05quantseq_workflow_revtime_quantseq05
Library Amplification
Step 4:
During the library amplification step sequences required
for cluster generation are introduced.
06quantseq_workflow_revtime_quantseq06
Library Amplification
Step 4:
Multiplexing can be performed with up to 9,216 barcode combinations
using the 96 available i7 indices and 96 i5 indices.
time_quantseq06
Sequencing
Step 5:
NGS reads are started at the very last nucleotide of the mRNA
using the Custom Sequencing Primer (CSP Version 2, included
in the kit) for sequencing. The reads generated during Read 1
reflect the cDNA sequence. With QuantSeq REV also paired-end
sequencing is possible.
08quantseq_workflow_rev

For viewing the whole workflow on page please click here

Featured Publications

Automation

autoQuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina

autoQuantSeq is the automated version of the QuantSeq 3’ mRNA-Seq Library Prep protocol in combination with its software. Hence, it features an automated all-in-one library preparation protocol designed to generate up to 9,216 Illumina-compatible libraries of the sequences close to the 3’ end of the polyadenylated RNA.

Automating the process of library preparation has the advantage of avoiding sample tracking errors, dramatically increasing throughput, and saving hands-on time.

QuantSeq protocol has been adapted for automated realization on the Sciclone NGS and Zephyr liquid handlers of PerkinElmer, the Hamilton Microlab STAR Workstations, the Agilent Bravo Automated Liquid Handling Platform and the Biomek platforms of Beckman Coulter. Contact us for implementation of QuantSeq on your automation platform.

Rapid Turnaround

Depending on the robot used the whole library preparation can be done in one day, including the manual preparation time. Since the individual protocol phases can be run on separate machines, further throughput enhancement can be achieved by parallelizing the workflow.

Easy Setup

An easy-to-follow Excel file guides you though preparation of all master-mixes and filling up the plates.

Flexibility of the Throughput

The QuantSeq kit is set up in a 96 well plate format and can generate libraries with up to 9,216 different barcode combinations. Depending on the liquid handler used you can either process any number of samples at the same time or need to run multiples of 8 reactions at once.

Avoiding Cross Contamination

The pre-PCR step and the post-PCR phase can be programmed on different machines which substantially reduces the risk of cross-contamination of the pre-PCR samples by the PCR products. 

FAQ

Frequently Asked Questions

Please find a list of the most frequently asked questions below. If you cannot find the answer to your question here or want to know more about our products, please contact support@lexogen.com.

Both kit versions yield sequences close to the 3’ end of transcripts. The difference is in the location of the Read 1 linker sequence. If it is located in the 5´ part of the second strand synthesis primer (QuantSeq Forward (FWD), Cat. No. 015), NGS reads will be generated towards the poly(A) tail (Figure 1). This version is recommended for gene expression analysis.

Figure 1 | Read orientation for QuantSeq FWD (Cat. No. 015).

With QuantSeq Reverse (REV, Cat. No. 016) the Read 1 linker sequence is located on the 5’ end of the oligodT primer and a Custom Sequencing Primer (CSP, included in the kit) is required for sequencing in order to start the read directly at the 3´ end (Figure 2). Based on this the exact 3’ UTR can be pinpointed.

QuantSeq_faq02

Figure 2 | Read orientation for QuantSeq REV with CSP (Cat. No. 016).

The upgraded protocol contains a more streamlined protocol with a shortened RNA removal step. Subsequent second strand synthesis and purification steps are also adjusted. Barcodes in the i7 Index Plate have been renamed (7001-7096, formerly BC01-96) and rearranged for a better nucleotide balance for sequencing when only few samples are multiplexed. With this new set-up all indices are now different from Illumina barcodes as former BC05 was replaced. With the QuantSeq FWD HT Kit (015.384) Lexogen furthermore provides a high-throughput version with optional dual indexing (i5 and i7 indices) allowing up to 384 barcode combinations (QuantSeq 3’ mRNA-Seq FWD HT User Guide).

For QuantSeq REV (Cat. No. 016) the Read 1 linker sequence is located at the 5’ end of the oligodT primer. Here a Custom Sequencing Primer (CSP Version 2, included in the kit) is required to achieve cluster calling on Illumina machines. It covers the poly(T) stretch and replaces the Multiplex Read 1 Sequencing primer.
PROVIDE THE RELEVANT INFORMATION TO YOUR SEQUENCING FACILITY ALONG WITH THE CSP!

HiSeq 2000, HiSeq 2500 (CSP Version 2 added on cBot)
CSP Version 2 should be provided in a tube strip at 0.5 µM final concentration in a volume of 120 µl (final concentration 0.5 µM, to be diluted in HT1 = Hybridization buffer). Take 0.6 µl of 100 µM CSP Version 2 and add 119.4 µl of HT1 buffer per sequencing lane. Place the 8-tube strip into the cBot position labeled primers.
HiSeq 2500 (CSP Version 2 replaces HP10 in cBot Cluster Generation Reagent Plate)
Alternatively, CSP Version 2 can be placed directly into the cBot Cluster Generation Reagent Plate. ATTENTION: The standard Illumina Multiplex Read 1 Sequencing Primer solution HP10 (for V4 chemistry located in row 2) provided in the cBot Cluster Generation Reagent Plate has to be REMOVED first! The Illumina V4 chemistry cBot Cluster Generation Reagent Plate only has 8 rows filled. A simple trick is to have the empty rows facing towards you, this way if you want to use a CSP in lane 1, you have to remove the HP10 solution from well 1 (first one on the far left) of the 2nd row, rinse the well a couple of times with HT1 and then add the diluted CSP Version 2. For this take 1.25 µl of 100 µM CSP Version 2 and add 248.75 µl of HT1 buffer per sequencing lane. The CSP should be at 0.5 µM final concentration in a volume of 250 µl (final concentration 0.5 µM, to be diluted in HT1 = Hybridization buffer). ATTENTION: Do not add the CSP to the Standard Illumina Multiplex Read 1 Sequencing Primer = HP10 solution! Always use fresh HT1 and add the CSP / HT1 dilution to the empty and rinsed well.

HiSeq 2500 – Rapid Run
Add 12.5 µl of 100 µM CSP Version 2 to 2487.5 µl HT1 = Hybridization buffer, resulting in a total volume of 2.5 ml and a final CSP concentration of 0.5 µM. In a rapid run, both lanes will use the same sequencing primer. It is not possible to run the two lanes with different sequencing primers.

MiSeq
Clustering is performed on the machine, not on the c-Bot. The MiSeq uses a reservoir of 600 µl with 0.5 µM sequencing primer final concentration, i.e., 3 µl of 100 µM CSP Version 2 in 597 µl HT1.

HiSeq 3000, HiSeq 4000 (CSP Version 2 replaces HP10 in cBot Cluster Generation Reagent Plate)
Usage of a custom sequencing primer is currently not supported on HiSeq 3000 and 4000 machines. A work around as described for the HiSeq2500 (CSP Version 2 REPLACES HP10 in the cBot Cluster Generation Reagent Plate) is possible though. ATTENTION: Do not add the CSP Version 2 to the HP10 solution! A primer mixture would result in low clusters calls and the resulting reads would be contaminated by poly(T) stretches. Always use fresh HT1 and add the CSP Version 2 / HT1 dilution to the empty and rinsed well.

The QuantSeq protocol is optimized for shorter reads (SR50, SR100) and yields mean library sizes of about 335 – 456 bp, with mean insert sizes of 203 – 324 bp. To generate longer libraries, use the QuantSeq-Flex First Strand Synthesis Module (Cat. No. 026), see QuantSeq 3’ mRNA-Seq User Guide, Appendix H, p.30.
The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is required. The amount of total RNA needed for QuantSeq depends on the poly(A) RNA content of the sample in question. This protocol was tested extensively with various cell cultures, animal and plant tissues, yeast, fungi, drosophila and human reference RNA. Typical inputs of 500 ng total RNA generate high quality libraries. For mRNA-rich tissues (such as kidney, liver, and brain) input material may be decreased to 50 ng without adjusting the protocol. However, for most efficient detection of low abundant transcripts RNA inputs from 500 ng to 200 ng are recommended.

Input RNA (UHR) Step 6: RNA Removal 95°C Step 16: PS Addition Library* Insert Library Yield PCR Cycles
 Start [bp]  End [bp] Mean Size* Mean Size ≥ 50 nt ≥ 100 nt ≥ 200 nt ng/μl nM
2000 ng 10 min 56 μl 132 2,000 456 324 97 % 80 % 31 % 2.0 10.2 11
500 ng 10 min 56 μl 132 2,000 364 232 98 % 78 % 27 % 1.8 9.8 12
100 ng 10 min 56 μl 132 2,000 350 218 97 % 74 % 21 % 2.1 11.3 14
50 ng 10 min 56 μl 132 2,000 389 257 96 % 70 % 20 % 2.4 12.7 15
10 ng 10 min 48 μl 132 2,000 350 218 96 % 70 % 24 % 2.6  14.1 18

*All libraries are prepared with external barcodes. Linker sequences are 122 bp including the 6 nt long external barcodes.

QuantSeq REV is only recommended for input amounts greater than 10 ng of total RNA. If you are limited in terms of input, we recommend using QuantSeq FWD. By choosing longer sequencing read lengths, you are also able to read into the 3’ ends.

For QuantSeq REV we recommend not to use input amounts lower than 10 ng total RNA. When using less than 10 ng of total RNA input please follow the recommendations indicated in the table below:

Protocol Step Standard Input (>10 ng) Low Input (10 ng) FFPE / Degraded RNA
Step 2 Incubate for 3 minutes at 85 °C, then cool to 42 °C.
Hold samples at 42 °C on the thermocycler.
Skip! Prepare pre-warmed FS1 / FS2 / E1 mastermix! Place RNA samples at room temperature.
Step 3 Prepare FS2 / E1 mastermix – pre-warm for 2 – 3 minutes at 42 °C. Prepare FS1 / FS2 / E1 mastermix – pre-warm for 2 – 3 minutes at 42 °C.
Step 4 Add pre-warmed mastermix to RNA / FS1 samples at 42 °C. Incubate for 15 minutes at 42 °C. Add pre-warmed mastermix to RNA samples at room temperature.
OPTIONAL: Increase incubation time to 1 hour at 42 °C.
Step 6 Incubate for 10 minutes at 95 °C. Incubate for 10 minutes at 95 °C.
Step 16 Add 56 μl of Purification Solution (PS). Reduce volume of Purification Solution (PS) to 48 μl.
Step 24 The qPCR assay is strongly recommended when processing samples with:
• Variable input amounts
• Variable RNA quality
• Different or new sample types (e.g., species, tissue, cell type)
The qPCR assay is strongly recommended for all low input, FFPE / degraded RNA library preps, to prevent over- or undercycling of the libraries.
Step 29 Add 30 μl of Purification Beads (PB). Reduce volume of Purification Beads (PB) to 27 μl.
The number of cycles for your endpoint PCR depends on the type of the RNA (tissue, organism), the RNA quality, and the RNA input amount. The reference values given in Appendix C, p.21 are based on Universal Human Reference RNA input and the mRNA content of other RNA sources might differ. To be on the safe side and to prevent under- or overcycling of your sample, we recommend performing a qPCR first. Therefore, we offer a PCR Add-on Kit for Illumina (020.96) with 96 additional PCR reactions. Use 5 µl of P7 Primer (7000) instead of an i7 index in step 27 of the single indexed PCR protocol. Dilute the double-stranded library from step 24 to 19 µl by adding 2 µl of Elution Buffer (EB) in order to have enough template for qPCR and endpoint PCR. Add 1.7 µl of the cDNA into a PCR reaction containing 7 µl PCR Mix, 5 µl P7 Primer 7000, 1 µl Enzyme Mix E from the PCR Add-on Kit, and SYBR Green I (or an equivalent fluorophore, to be provided by the user) to a final concentration of 0.1x (diluted in DMSO). Make the total reaction volume up to 30 µl with EB. Conduct at least 35 cycles to make sure the amplification reaches the plateau. Afterwards take the fluorescence value where the plateau is reached and calculate where the fluorescence is at 50 % of the maximum (see Fig. 3). The value where the fluorescence reaches the maximum (plateau) is taken (15388) and the fluorescence at 50 % of this values (7694) shows which cycle number is optimal for the endpoint PCRs. For the sample in Fig. 3 this would be 15 cycles when using 1/10th of your sample. If the optimal cycle number lies within two values, it is recommended to always round up to the higher number in order to get more yield. As in the endpoint PCR 10x more cDNA will be used compared to the qPCR, three cycles can be subtracted from the determined cycle number, hence in this example 12 cycles should be used for the endpoint PCR. This is the cycle number you should use for the endpoint PCR using the remaining 17 µl of the template.Calculation_of_the_number_of_cycle_for_the_endpoint_PCR

Figure 3: Calculation of the number of cycles for the endpoint PCR

Once the number of cycles for the endpoint PCR is established for one type of sample, you can use it in the following experiments. For higher yields you can increase the fluorescence level of the endpoint PCR up to 80 % without overcycling your sample.

In the table below you can see some recommended cycle numbers for the endpoint PCR using 500 ng total RNA input of different RNA sources.

Input RNA (500 ng) Cycles ng/µl nM
UHRR 12 1.8 9.8
HBRR 13 1.9 10.5
M.m. heart 13 3.8 20.9
M.m. brain 13 2.9 15.6
M.m. liver 12 1.3 6.7
M.m. kidney 12 2.3 12.2
M.m. spleen 13 1.4 8.0
M.m. lung 14 2.6 15.5
M.m. embryonic stem cells 11 1.3 7.5
M.m. myoblast 12 0.9 5.2
M.m. fibroblast 14 1.0 5.6
M.m. myoblast progenitors 11 2.1 11.5
M.m. neural progenitors 12 1.2 7.0
Arapidopsis th. 13 1.7 9.8
Tomato seeds 16 1.7 9.4
Fungi RNA 13 1.24 7.1
Yeast RNA (S.c.) 12 1.2 7.7
Drosophila melanogaster 13 1.6 7.9

Yes, low quality and FFPE samples can be used with QuantSeq. Some minor protocol modifications are required though. These recommendations are indicated in the table below:

Protocol Step Standard Input (>10 ng) Low Input (10 ng) FFPE / Degraded RNA
Step 2 Incubate for 3 minutes at 85 °C, then cool to 42 °C.
Hold samples at 42 °C on the thermocycler.
Skip! Prepare pre-warmed FS1 / FS2 / E1 mastermix! Place RNA samples at room temperature.
Step 3 Prepare FS2 / E1 mastermix – pre-warm for 2 – 3 minutes at 42 °C. Prepare FS1 / FS2 / E1 mastermix – pre-warm for 2 – 3 minutes at 42 °C.
Step 4 Add pre-warmed mastermix to RNA / FS1 samples at 42 °C. Incubate for 15 minutes at 42 °C. Add pre-warmed mastermix to RNA samples at room temperature.
OPTIONAL: Increase incubation time to 1 hour at 42 °C.
Step 6 Incubate for 10 minutes at 95 °C. Incubate for 10 minutes at 95 °C.
Step 16 Add 56 μl of Purification Solution (PS). Reduce volume of Purification Solution (PS) to 48 μl.
Step 24 The qPCR assay is strongly recommended when processing samples with:
• Variable input amounts
• Variable RNA quality
• Different or new sample types (e.g., species, tissue, cell type)
The qPCR assay is strongly recommended for all low input, FFPE / degraded RNA library preps, to prevent over- or undercycling of the libraries.
Step 29 Add 30 μl of Purification Beads (PB). Reduce volume of Purification Beads (PB) to 27 μl.

The quality of RNA from FFPE tissues can vary greatly. We recommend measuring the DV200 value (the percentage of RNA greater than 200 nt in length) in addition to RIN values, as RIN values become less meaningful for highly degraded samples.

Libraries prepared from FFPE RNA input typically require different (often higher) PCR cycle numbers than those prepared with high quality RNA input (see table below for examples). The DV200 value is also not always a reliable predictor of the required number of PCR cycles needed.

When preparing libraries for comparative gene expression profiling, all libraries that will eventually be compared should be amplified using the same number of PCR cycles. To prevent under- or overcycling the libraries, we therefore strongly recommend performing a qPCR assay to determine the optimal number of PCR cycles for the set of samples to be processed within each experiment.

ng FFPE RNA* Input PCR Cycle Number
50 ng FFPE 15
10 ng FFPE 18

* Please be aware the values in the table are guidelines only and are based on Mm brain FFPE RNA with a RIN of 1.8 (DV200 of 51 %). For different sources of RNA, and variable RNA qualities, more (or less) PCR cycles might be needed.

If there are a range of optimal PCR cycle numbers predicted for your samples, you may wish to perform an additional endpoint PCR test to check the library yields are sufficient for sequencing. This is ideally done using half the library volume for a couple of samples that have different cycle number predictions (e.g. lowest, middle, and highest cycle number), and using the average number of cycles for the endpoint PCR (adding one additional cycle to account for using half the library volume). After purifying and quantifying these libraries, you can evaluate the relative yields and adjust the number of PCR cycles accordingly. Please note additional PCR and purification reagents provided in the PCR Add-on Kit for Illumina (Cat. No. 020.96) and the Purification Module with Magnetic Beads (Cat. No. 022.96) would be required for this type of endpoint PCR testing.

Lexogen’s QuantSeq kit is a library preparation protocol designed to generate sequence-ready Illumina-compatible libraries from polyadenylated RNA within 4.5 hours. When carrying out the protocol for the first time, please allow for more time and read the entire User Guide first.
QuantSeq libraries are intended for a high degree of multiplexing. Barcodes are introduced as standard external barcodes during the PCR amplification step. With the up to 96 external i7 barcodes (i7 Index Plate, 7001-7096) included in the kit and the additionally available four external i5 indices (5001-5096, i5 Dual Indexing Add-on Kit, Cat. No. 047), up to 9,216 samples can be multiplexed and sequenced per lane on an Illumina flow cell.

ATTENTION: QuantSeq REV cannot be multiplexed with other library preps as the Custom Sequencing Primer is needed for sequencing.

With QuantSeq REV and the Custom Sequencing Primer it is possible to exactly pinpoint the 3’ end during Read 1. The reads generated during Read 1 reflect the cDNA sequence, and are therefore in a strand orientation opposite to the genomic reference.
For QuantSeq REV (Cat. No. 016) we do not recommend using TopHat2, since there is hardly any need to search for junctions. Nearly all sequences will originate from the last exon and the 3’untranslated region (UTR). In case of no detected junction, TopHat2 may run into difficulties. Hence, Bowtie2 or BWA can be used for mapping in this case.
More information on the data analysis can be found here.
While single read sequencing does not require any trimming using QuantSeq REV (Cat. No. 016), paired-end sequencing may require the first 12 nucleotides of Read 2 to be trimmed. Alternatively, a less stringent aligner STAR Aligner could be used with the number of allowed mismatches being set to 16 for paired-end reads.
In case of adapter contamination detection it is crucial to trim these sequences (e.g., cutadapt, trim-gallore, or bbduk) in order to align the reads
The QuantSeq 3’ mRNA-Seq REV kit is appropriate for HiSeq 2000/2500, HiSeq 3000, HiSeq 4000, GAIIX, and MiSeq. We do not recommend to sequence QuantSeq REV libraries on NextSeq 500 or NextSeq 550 Illumina platforms. The polyadenylation signal located at the 3’ end, which is quite similar between transcripts, will result in a reduced complexity at the beginning of the reads leading to a lower sequencing quality.
Single-read 50 (SR50) sequencing runs are suitable for both QuantSeq versions. Although, for QuantSeq REV it is favorable to use longer runs (SR100), since starting the read exactly at the 3`UTR will also show the polyadenylation signal, which covers up to 25 nt from the transcription end. As the polyadenylation signal is quite similar for all transcripts, it is better to have longer reads >SR50 available for mapping in order to increase the number of uniquely mapping reads.
If you want to determine the optimal number of cycles for your endpoint PCR using dual indexing, you can still use the PCR Add-on Kit according to the instructions (see FAQ 1.7). The single indexing PCR (i7 only) of the PCR Add-on Kit and the dual indexing PCR (i5 and i7) run with the same efficiency, hence there is no need to exchange any solutions.
For QuantSeq REV it is essential to use the CSP (provided with the QuantSeq kit) for Read 1 sequencing. Make sure that your sequencing facility is aware of this. When handing in QuantSeq REV libraries for sequencing, please include the CSP and the information on how to use the CSP. The CSP should never be mixed together with the standard Illumina Read 1 Sequencing primer. A primer mixture would result in low clusters calls and the resulting reads would be contaminated by poly(T) stretches.
Transcripts may have different and not yet annotated 3’ ends, which might be mistaken for internal priming events of the oligodT primer, when in fact those are true 3’ ends. Artificial spike-in transcripts such as the SIRVs or the ERCC spike-in transcripts only have one defined 3’ end, this provides the only true measure to determine internal priming. If true internal priming is detected, this could be a result of mis-priming during reverse transcription, for instance if the temperature before or during reverse transcription was too low. As outlined in the general section of the User Guide, unless explicitly mentioned, all steps should be carried out at room temperature between 20 °C and 25 °C. In particular, for First Strand cDNA Synthesis (p.11): Do not cool the FS2/E1 mastermix (step 3) and have the RNA/FS1 samples at 42 °C when adding the FS2/E1 mastermix (step 4) to avoid mishybridization. Mix properly by pipetting. Do not forget to shortly spin down the samples at room temperature before and after adding the FS2/E1 mastermix. Centrifugation should not be carried out at 4 °C, always spin down at room temperature! Raising the reaction temperature to 50 °C for reverse transcription can be used for additional prevention of mis-priming.
  • First Strand cDNA Synthesis (p.11-12):
    1. At step 3 pre-warm the FS2 / E1 mastermix for 2 – 3 minutes at 42 °C while the RNA / FS1 samples are denaturing for 3 minutes at 85 °C – Do not cool the mastermix on ice!
    2. After the RNA / FS1 samples have cooled to 42 °C, spin these down briefly and then immediately return to the thermocycler and hold at 42 °C.
    3. Add the pre-warmed FS2 / E1 mastermix to the RNA / FS1 samples on the thermocycler at 42 °C (step 4) and mix properly. Any drop in temperature at this point can cause mishybridization! Seal the plate or tubes and begin the 42 °C incubation.

    NOTE! Spin down the samples at room temperature before and after adding the FS2 / E1 mastermix.

  • If step is skipped (low input or degraded samples i.e. ≤10 ng, or FFPE samples):
    1. Prepare your RNA samples in 5 μl volumes.
    2. Prepare a mastermix containing 5 μl FS1, 9.5 μl FS2, and 0.5 μl E1, mix well, spin down, and pre-warm at 42 °C on a thermocycler for 2 – 3 minutes.
    3. Bring your RNA samples to room temperature while the mastermix is pre-warming.
    4. Spin down the pre-warmed FS1 / FS2 / E1 mastermix and add 15 μl to each RNA sample. Quickly mix, seal the plate or strip-tubes, spin down briefly at room temperature, and then commence the 42 °C incubation for 15 minutes (or 1 hour for low input RNA (≤ 10 ng)).
    1.28

  • Proceed immediately to the RNA removal after the reverse transcription is complete! (step 4-5). The previous safe stopping point after reverse transcription (step 4) has been removed. Do not place the samples on ice, and do not store samples at -20 °C at this point! Cooling the samples below room temperature at this point can cause mishybridisation! Best practice handling would be as follows:
    1. After the 42 °C incubation is complete spin down the plate/tubes briefly and place at room temperature.
    2. Immediately add the RNA Removal Solution (RS, thawed at room temperature) to the samples, mix well.
    3. Briefly spin down the plate / tubes at room temperature, then place on the thermocycler to commence the 10 minute incubation at 95 °C (step 6).

    NOTE! To minimise temperature drops at this point the reactions can also be kept at 42 °C while the RNA Removal Solution (RS) is added: Briefly spin down the samples after step 4 and place them back on the thermocycler at 42 °C, remove the sealing foil / tube caps, add the RNA Removal solution to the samples, mix, re-seal the plate / tubes, quickly spin down, and place back on the thermocycler block and re-start the program for the 95 °C incubation.

A second peak between 1000–9000 bp is an indication of overcycling. The library prep has been very efficient and a lot of cDNA was generated. Hence, the PCR ran out of primers and template started to denature and reanneal improperly. This results in longer, bulky molecules that migrate at a lower speed on the Bioanalyzer chip or gels. This can interfere with exact library quantification if relying solely on the Bioanalyzer results. Therefore, a qPCR assay for exact library quantification should be used additionally if such a high molecular weight peak occurs.
For future QuantSeq library preps on similar samples reduce your PCR cycle number accordingly to prevent overcycling. Overcycling may lead to a distortion in gene expression quantification and hence should be avoided.
A carryover of Purification Beads (PB) results in a peak around and beyond the upper marker of the Bioanalyzer. Make sure not to transfer any beads after the final elution in step 41 or 42 for single or dual indexing PCR respectively. Leave approximately 2 µl of the eluate on the beads and do not try to transfer the complete sample, as this will lead to bead carryover. Put your samples once again on the magnet and incubate for 5 minutes. Transfer 15-17 μl of the supernatant into a fresh PCR plate.
The PCR Add-on Kit for Illumina (Cat. No. 020) includes a Reamplification Primer that can be used to add some PCR cycles for your undercycled libraries (QuantSeq 3’ mRNA-Seq User Guide, Appendix F: Library Reamplification, p.27). In general, as QuantSeq is intended for a high degree of multiplexing, undercycled libraries can still be used for preparing a lane mix. The lane mix may need to be concentrated if many libraries of the lane mix were undercycled. Please note that currently only single indexed libraries can be reamplified. If you need to reamplify dual indexed libraries, please contact info@lexogen.com.
Universal Human Reference RNA (UHRR, Agilent) is a good positive control, most of the reference values given in the User Guide are also based on UHRR input.
For PE sequencing use QuantSeq REV (Cat. No. 016). We do not recommend paired-end sequencing for QuantSeq FWD (Cat. No. 015), as the quality of Read 2 would be very low due to the poly(T) stretch at the beginning of Read 2.
The loading amounts indicated below are provided a guideline for sequencing QuantSeq FWD/REV libraries in pure pools for each instrument. Multiplexing QuantSeq with other external library types is not recommended (see FAQ 1.33). We therefore cannot provide optimal pooling or loading amount guidelines for lanemixes that include QuantSeq libraries in diverse multiplexing setups.
Loading amounts may still need to be adjusted to obtain optimal cluster densities for your respective instrument, or if sequencing chemistries are changed. Inaccuracies in library quantification can affect the loading amount required. Please ensure accurate quantification of lane mixes and lane mix dilutions when preparing for loading.
Unless otherwise indicated the values given in the table below are based on customer experiences. For further inquiries regarding loading amounts please contact info@lexogen.com.

Sequencer QuantSeq FWD (Cat. No. 015) QuantSeq FWD with UMIs QuantSeq REV (Cat. No. 016)
HiSeq 3000 / HiSeq 4000 / HiSeqXTen 280 – 350 pM 1 ** **
HiSeq 2000 / HiSeq 2500 10 pM ** 10 – 12.5 pM
MiSeq 6 – 15 pM ** 6 – 15 pM
MiniSeq 1.3 – 1.8 pM ** **
NextSeq 500 / NextSeq 550 2 – 2.5 pM 2 1.8 pM **
NovaSeq 6000 Standard: 400 – 500 pM 3 , Xp: 325 – 400 pM 4 ** **
PhiX spike-in % 1 – 5 % 15 – 30 % Not recommended *

* PhiX cannot be sequenced in QuantSeq REV runs due to the use of a custom sequencing primer which does not bind to PhiX.
** Please inquire at info@lexogen.com.
1 Loading amounts are based on customer-supplied feedback for optimal sequencing quality on HiSeq 4000.
2 The values for NextSeq 500 / 550 are recommended loading amounts and have been evaluated by Lexogen to ensure optimal cluster densities of 200 – 260 K/mm2 are achieved. We have determined that loading amounts below 2 pM may result in lower cluster densities and reduced index read quality.
3 Loading amounts for NovaSeq Standard workflow are based on user experience using S2 flow cells, and should be relevant for all flow cell types. This range is further calculated according to Illumina’s recommendation to use 1.5x the loading concentrations for HiSeq 4000. The calculated loading amounts shown fall within the Illumina recommended range for PCR-amplified library pools (300 – 600 pM).
4 Loading amounts for NovaSeq Xp workflow are based on customer-supplied feedback from sequencing of SENSE mRNA V2 libraries. These concentrations also relate to customer-validated loading amounts used for HiSeq 4000 instruments.

In general, we recommend processing a minimum of 8 samples, using a complete set of eight i7 indices for multiplexing (e.g., 7001-7008). However, if fewer barcodes are required care should be taken to always use indices which give a well balanced signal in both lasers (red and green channels) for each nucleotide position. All columns (1-12) and rows (A-H) fulfill these criteria. The individual libraries within a lane should be mixed at an equimolar ratio to ensure this balance. An evaluation tool to check the color balance of index subsets is available under Support Tools.
Yes! All QuantSeq FWD and REV kits ordered after November 2016 are supplied with a code to access the QuantSeq data analysis pipelines on the Bluebee® Genomics Platform.

NOTE! The QuantSeq “FWD” and “REV” pipelines are specifically designed for libraries prepared with QuantSeq FWD 3’ mRNA-Seq Library Prep Kit (Cat. No. 015) or the QuantSeq REV 3′ mRNA-Seq Library Prep Kit (Cat. No. 016), respectively and the activation codes provided with each kit lead you to the respective pipeline only.

Analysis pipelines for QuantSeq (FWD, FWD-UMI, and REV) are currently available for the following species:

Common name (if available) (Species)

  • African Oil Palm (Elaeis guineensis)
  • Arabidopsis Thale cress (Arabidopsis thaliana)
  • Baker’s Yeast (Saccharomyces cerevisiae)
  • Brain-Eating Amoeba (Naegleria fowleri)
  • Chicken (Gallus gallus)
  • Cow (Bos taurus)
  • Daphnia (Daphnia pulex)
  • Dog (Canis lupus familiaris)
  • Drummond’s Rockcress (Boechera stricta)
  • Ferret (Mustela putorius furo)
  • Fruit Fly (Drosophila melanogaster)
  • Fungus (Fusarium oxysporum)
  • Goat (Capra hircus)
  • Human (Homo sapiens)
  • Melon Fly (Bactrocera cucurbitae)
  • Mouse (Mus musculus)
  • Nematode Roundworm (Caenorhabditis elegans)
  • Painted Turtle (Chrysemys picta bellii)
  • Pig (Sus scrofa)
  • Poplar (Populus poplar)
  • Potato (Solanum tuberosum)
  • Purple False Brome (Brachypodium distachyon)
  • Rabbit (Oryctolagus cuniculus)
  • Rat (Rattus norvegicus)
  • Rice (Oryza sativa)
  • Salmon (Salmon salar)
  • Sorghum (Sorghum bicolor)
  • Starlet Sea Anemone (Nematostella vectensis)
  • Tomato (Solanum lycopersicum)
  • Yeast (Candida albicans)
  • Yeast (Kluyveromyces lactis)
  • Zebrafish (Danio rerio)

New species can be added upon request. Please contact bioinfo@lexogen.com if your species of interest is not listed.

All QuantSeq FWD and REV kits ordered after November 2016 are supplied with an activation code to access the QuantSeq Data Analysis pipelines on the Bluebee® Genomics Platform.

The activation codes are printed on a sticker that is attached to the side of the small reagent box (stored at -20 °C) inside the main QuantSeq Kit box (see Figure 4). Activation codes for additional runs can be purchased from Lexogen via the webshop. Activation codes registered after September 20, 2018 are valid for two years. The input file size is limited to 1.5 GB per fastq(.gz) file. If you have larger input files or for further inquiries, please contact info@lexogen.com.

Bluebee_Sticker_Location
Figure 4 | Location of the Bluebee® Data Analysis activation code on QuantSeq Kit reagent boxes.

The count summary files from the results of the data analysis pipeline on Bluebee® contain mapped read counts for Ensembl gene IDs. Ensembl gene IDs can be converted into other gene ID types, or additional annotations added using the BioDBnet tools: https://biodbnet-abcc.ncifcrf.gov/db/db2db.php. Simply input the list of Ensembl gene IDs, select the input format as “Ensembl Gene ID”, and then select the output format(s) desired.
The UMI Second Strand Synthesis Module (Cat. No. 081.96) is only compatible with QuantSeq FWD Library Prep. This is because the UMI-containing random primers in this module contain the partial p5 adapter sequence, which is the same as the partial adapter included on the oligo(dT) primers used for QuantSeq REV Library Prep. Hence, using the UMI Module with QuantSeq REV will not result in an amplifiable library. If you are interested in including Unique Molecular Identifiers for QuantSeq REV Library Prep please contact us at info@lexogen.com.
Yes, the i5 Dual Indexing Add-on Kits can be used for dual indexing of QuantSeq REV libraries. Please refer to the i5 Dual Indexing Add-on Kits Instruction Manual (047IM109) for details of dual-indexed library amplification and purification.
For further information about the i5 Dual Indexing Add-on Kit for QuantSeq/SENSE please see the QuantSeq REV online FAQs 4.1 onwards.
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 or SENSE 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).

The autoQuantSeq 3′ mRNA-Seq protocol is currently automated on the following liquid handling instruments:

    • Perkin Elmer: Sciclone® / Zephyr®
    • Hamilton: Microlab STAR / STARlet
    • Agilent: NGS Workstation (NGS Bravo Option B)
    • Beckman Coulter: Biomek FXP, and Biomek i7
    • Eppendorf: EpMotion® 5075

More information about QuantSeq Automation on Agilent Bravo can be found in our QuantSeq Automation Application Note.

More information about QuantSeq Automation on Beckman Biomek can be found in the Application Overview Flyer.

Please contact us if you are interested in automating QuantSeq on additional liquid handling platforms.

Script files for QuantSeq Automated protocols are available for download from our sftp server. Please contact info@lexogen.com for the login details.

The protocol for the Hamilton STAR is designed to process any number of samples from 1 to 48 and the Beckman Coulter Biomek from 1 to 96.

For the PerkinElmer Sciclone any number of columns from 1 to 12 can be run. The machine will always take the barcodes from the first N wells of the barcode plate, starting with column 1. Therefore, you can run an assay with less than 96 (for example 24) reactions in multiples of 8. The program for Agilent´s Bravo also runs multiples of 8 reactions.

Unfortunately no. The Phase1-PrePCR of the autoSENSE protocol is currently only available for the Sciclone NGS workstation.
The protocol is intended and programmed to run on this variant of the Sciclone liquid handler but this configuration has not been tested. Therefore, we recommend contacting Lexogen for on-site support with protocol installation.
Yes. The protocol is implemented in a deck format compatible with both STAR and STARlet versions of the Hamilton Microlab liquid handler series.
The use of the Master Plate is recommended for high-throughput processing. It needs to be filled in manually by the operator in order to have the machine distributing the reagents to the individual microplates. Without the Master Plate, the operator has to fill all microplates manually.
On the PerkinElmer Sciclone one can choose to do the thermal treatment off-deck (using an external thermocycler) or on-deck (using built-in thermolocators). This option is up to the user. It has to be set in the code and needs to be selected at the installation of the protocol. Both options require manual interventions. If a stand-alone thermocycler is available in a comfortable vicinity of the robot, we suggest to use off-deck thermal treatment, in particular because a stand-alone thermocycler has a heated lid preventing condensation.
For the Hamilton STAR and Agilent Bravo only an off-deck option is available, while the Beckman Coulter Biomek is a fully walk-away protocol with on-deck thermal treatment, including the PCR reaction.
To use the protocol, you also need a specific hardware deck setup and a software setup, which make your liquid handler adapted to NGS library preparation protocols. This adaptation is done by a Hamilton application specialist, typically by installing Hamilton’s NEBNext Ultra Library Prep Kit for Illumina (E7370) protocol. Once this is done, installing the autoQuantSeq protocol is simple and can be done by the user.
Yes, if thermal treatments are run off-deck, five such interventions (including PCR) are required. The machine stops and asks the operator to do various steps such as, e.g., take the plate with samples, seal it with film, do the thermal step on the external thermocycler, unseal the plate, place it on deck again, and resume the run.
The Excel protocol workbook will automatically calculate the times when the interventions are due. Additionally, during the run, the dialog window prompting the operator for intervention also shows a message like ‘Next intervention follows in … minutes’.
To simplify the manual preparation, the Elution Buffer (EB) is presented in a 50 ml reservoir (trough). Robotic aspiration from this type of vessel requires a higher dead volume. Therefore, the amount of the EB required for automation might exceed the amount delivered in the kit. If this is the case, the EB can simply be substituted by 10 mM TRIS pH 8.0.
The QuantSeq 3’ mRNA-Seq kit contains enough components to generate 24, 96 or 2×96 library preps manually. There will be sufficient reagent to run all reactions at once on your liquid handler, however, if you want to split your kit into several machine runs you might loose a few reactions due to the higher dead volume needed compared to the manual preparation.

The Globin Block Modules can only be used with the QuantSeq 3’ mRNA-Seq Kits. They are compatible with both the FWD (Cat. No. 015) and REV (Cat. No. 016) versions of the kit and can also be used with the kits for Ion Torrent (Cat. No. 012). For information on the use of Globin Block Modules with the QuantSeq 3’ mRNA-Seq Library Prep Kit for Ion Torrent protocol, please contact info@lexogen.com.

The Modules are not compatible with the SENSE mRNA-Seq V2 (Cat. No. 001) or SENSE Total RNA-Seq (Cat. No. 009) Library Prep Kits.

NOTE: The modules are not intended for use with any other external library prep protocols.

Yes. When using the modules with the older version of the QuantSeq Kits, the RS-Globin Block solution replaces the RNA Removal Solution 1 (RS1) at step 5. After this, follow the protocol as written, proceeding with the incubation in step 6, and the addition of the RNA Removal Solution 2 (RS2) in step 7.
Currently Globin Block Modules are available for human (Homo sapiens) and pig (Sus scrofa). These contain species-specific globin blockers. The cross-reactivity of these solutions for globin reduction in other species has not been evaluated. If you are interested in globin block solutions for additional species please contact us at info@lexogen.com and tell us which species you are interested in.
Yes, the Globin Block Modules can also be used to prepare QuantSeq libraries from low quality total RNA input from blood (RIN <6). This will also result in a reduction of reads mapping to globin genes. Please refer to the protocol modifications for low quality samples given in the Globin Block Modules for QuantSeq Instruction Manual.
If you are interested in using lower input amounts (<50 ng) of blood total RNA, please contact us at info@lexogen.com for further information on protocol modifications.
We strongly recommend performing a qPCR assay to determine the optimal number of PCR cycles for the library amplification. Information on the qPCR assay can be found in the QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina User Guide (Appendix E, p.25). The PCR Add-on Kit for Illumina (Cat. No. 020.96) includes PCR and enzyme mix reagents and primers required for the qPCR assay. In addition, SYBR Green I Nucleic Acid Gel Stain (10,000X in DMSO, Sigma-Aldrich Cat. No. S9430) is also required (user provided).

Typically, libraries prepared from blood total RNA using Globin Block require one additional PCR cycle compared to libraries made with the standard QuantSeq Kit reagents. The table below provides some example PCR cycle numbers for different input amounts.

ATTENTION! This table should be interpreted as a guideline only! Blood samples can be highly variable depending on origin and quality. We strongly recommend performing the qPCR assay to determine cycle numbers directly.

Species Whole Blood or
Leukocyte Blood
Input Amount PCR Cycles
 Undepleted  + RS-Globin Block
Human Whole blood 250 ng 13 14
50 ng 15 16
Leukocyte-enriched blood 50 ng 16 17
50 ng * 15 16
Pig Whole blood 100 ng 16 17

*Indicates RNA isolated using PAXgene® Blood System (Tubes and Kit, Qiagen), which includes 24-hour red blood cell lysis. All other RNA was extracted using the SPLIT RNA Extraction Kit (Cat. No. 008.48). All libraries were prepared with single indexing. Linker sequences are 122 bp including 6 nt long i7 indices.

Libraries prepared from human blood RNA with standard QuantSeq Library Prep Kit and protocol display distinct peaks in bioanalyzer traces at 197 bp, 212 bp, 222 bp, 235 bp, and 312 bp, which correspond to abundant globin mRNA library fragments (HBA1, HBA2, and HBB). These peaks are reduced in libraries prepared with the RS-Globin Block, Homo sapiens module (RS-GBHs), as shown below in Figure 1 (50 ng whole blood total RNA).
Globin Block libraries can also display: a longer library size distribution, and/or, peaks present at larger molecular weights (~400 bp and higher).
FAQ_Figure1_GlobinBlock

Figure 1 | Bioanalyzer traces for human QuantSeq FWD libraries prepared with (+Globin Block) and without (Standard) the RS-Globin Block solution (RS-GBHs, Cat. No. 070.96). Replicate libraries were prepared from 50 ng of whole blood (WB) RNA with the Standard QuantSeq FWD protocol (blue and red traces), versus QuantSeq +Globin Block (green and turquoise traces). RNA was isolated using the SPLIT RNA Extraction Kit without red blood cell lysis (Lexogen). Grey arrows indicate major globin peaks reduced in +Globin Block Libraries.

Pig blood libraries prepared with RS-Globin Block, Sus scrofa (RS-GBSs) also show an altered peak profile compared to libraries prepared with standard QuantSeq RS (see Figure 2 below).

FAQ_Figure2_GlobinBlock
Figure 2 | Bioanalyzer traces for pig QuantSeq FWD libraries prepared with (+Globin Block) and without (Standard) the RS-Globin Block solution (RS-GBSs, Cat. No. 071.96). Replicate libraries were prepared from 100 ng of whole blood (WB) RNA with the Standard QuantSeq FWD protocol (blue and red traces), versus QuantSeq +Globin Block (green and turquoise traces). RNA was isolated using the Preserved Blood RNA Purification Kit + Dnase I Kit (Norgen Biotek). Grey arrows indicate major globin peaks reduced in +Globin Block Libraries.

Globin reduction is best evaluated prior to sequencing by preparing control libraries from the same RNA sample using the standard QuantSeq protocol, alongside libraries prepared with QuantSeq and RS-Globin Block.

The percentage of reads mapping to globin mRNAs is calculated as the fraction of read counts uniquely mapping to globin genes divided by the total uniquely mapped reads.
Uniquely mapped read counts are found in the read_counts.txt files given as output from the Data Analysis pipeline on the Bluebee® Genomics Platform.
Lists of Ensembl gene IDs for human and pig globin genes are provided in the tables below.
For further information on data analysis for QuantSeq libraries prepared with Globin Block Modules please contact info@lexogen.com.

Table 1. Ensembl Gene IDs for human globin genes.

Gene Symbol Ensembl Gene ID
HBQ1 ENSG00000086506
HBZ ENSG00000130656
HBA2 ENSG00000188536
HBG2 ENSG00000196565
HBA1 ENSG00000206172
HBM ENSG00000206177
HBZP1 ENSG00000206178
HBE1 ENSG00000213931
HBG1 ENSG00000213934
HBD ENSG00000223609
HBBP1 ENSG00000229988
HBB ENSG00000244734

Table 2. Ensembl Gene IDs for pig (Sus scrofa) globin genes.

Gene Symbol Ensembl Gene ID
HBZ ENSSSCG00000007975
HBM ENSSSCG00000007977
HBE1 ENSSSCG00000014726
HBB ENSSSCG00000014725
HBA ENSSSCG00000007978

The i5 Unique Dual Indexing Add-on Kit for QuantSeq/SENSE (5001 – 5096) provides a 96-well plate containing 96 unique i5 indices (Cat. No. 047.96). This kit is designed for unique dual indexing in combination with Lexogen’s 96 i7 indices (included in all QuantSeq 3’, QuantSeq-Flex, and SENSE library prep kits (Cat. No. 001, 009, 015, 016, 033, 034, ,035, and 042; and separately: Cat. No. 044.96)). Up to 96 uniquely dual-indexed libraries can be prepared for sequencing in a single lane or run.

The i5 Dual Indexing Add-on Kit for QuantSeq/SENSE (5001 – 5004, Cat. No. 047.4) provides four different i5 index primers (5001 – 5004). Each tube contains sufficient volume for preparing 24 (Cat. No. 047.4×24), or 96 (Cat. No. 047.4×96) libraries. This kit is therefore ideal for combinatorial dual indexing strategies (see FAQ 4.6). In combination with 96 i7 indices a maximum of 384 (4 i5 x 96 i7) dual-indexed libraries with unique i5 / i7 index combinations can be multiplexed in a single sequencing lane or run.

These i5 Dual Indexing Add-on Kits are compatible with all QuantSeq and SENSE Library Prep Kits for Illumina, specifically:

      • QuantSeq 3’ mRNA-Seq Library Prep Kits (FWD and REV) for Illumina (Cat. No. 015 and 016)
      • QuantSeq-Flex Targeted RNA-Seq Library Prep Kits V2 for Illumina (Cat. No. 033, 034, and 035)
      • SENSE mRNA-Seq Library Prep Kit V2 for Illumina (Cat. No. 001)
      • SENSE Total RNA-Seq Library Prep Kit for Illumina (Cat. No. 009, 042)

These add-on kits are not compatible with the Small RNA-Seq Library Prep Kit for Illumina (Cat. No. 052, 058). This is because the internal adapters in the small RNA libraries differ from those used in QuantSeq and SENSE libraries.

The Dual PCR Mix (Dual PCR, purple cap) must be used instead of the standard PCR Mix (PCR, yellow cap for QuantSeq and SENSE mRNA-Seq Kits, white cap for SENSE Total RNA-Seq Kits), for amplification of dual-indexed libraries. The Dual PCR Mix contains no primers, which enables the i5 and i7 index primers of your choice to be added to each library, in order to prepare libraries with unique i5 / i7 index combinations. If the standard PCR Mix (yellow/white cap) is used for preparing dual indexed libraries together with i5 and i7 index primers, there will be a fraction of insert fragments in the final library that will not contain any i5 index. Therefore, reads will be lost during demultiplexing.
The i5 and i7 indices are added during the final library amplification. Please follow the detailed protocol for Endpoint PCR and Post-PCR Purification steps for dual-indexed library preparation in the Instruction Manual for the i5 Dual Indexing Add-on Kits (047IM109). Differences to the protocol for amplification of single-indexed QuantSeq libraries are as follows:

      • The Dual PCR Mix (purple cap) must be used instead of the standard PCR Mix (yellow/white cap).
      • The total volume of the PCR is 35 ul.
      • The volume of Purification Beads (PB) to add at step 6 is also 35 ul (or 31.5 ul for libraries generated from low input or degraded / FFPE RNA samples).

The Dual PCR Mix is provided in the i5 Dual Indexing Add-on Kits along with the i5 index primers. The i7 index primers and Enzyme Mix (E3) are provided in the QuantSeq Kits (Cat. No. 015, 016, 033, 034, and 035). The Enzyme Mix (E), from the PCR Add-on Kit for Illumina (Cat. No. 020.96) can also be used for this endpoint PCR. The purification reagents for Post-PCR Purification are also provided in the standard QuantSeq Kits.

The i5 index sequence to enter into Illumina sample sheets must be in forward (i5) or reverse complement (i5rc), depending on the instrument and flow cell type used for the sequencing run. The i5 sequences in both orientations are provided for download in the Lexogen i7 and i5 Index Sequences excel file, available from the QuantSeq and SENSE product pages, and Index Balance Checker Support Tools page.
All i5 and i7 indices are 6 nt long, however if you are running 8 cycle index reads, then the first 2 nucleotides from the adjacent adapter sequence need to be added to the sequence in the sample sheet. These are: AC for i5 (forward orientation), and GT for i5rc (reverse complement orientation).
Dual indexing in general, which uses both i7 (Index 1) and i5 (Index 2) indices, enables multiple RNA-Seq libraries to be multiplexed for a single sequencing lane or run. Using dual indexing, the unique combination of both i5 and i7 indices determines the sample identification, which increases the accuracy of read identification when the sequencing data is demultiplexed.
There are two types of dual indexing: combinatorial dual indexing, and unique dual indexing (Fig. 1).

i5-dual-indexing-strategies
Figure 1 | Comparison of combinatorial and unique dual indexing strategies. Regardless of the strategy used, each library (A – F) must have a unique combination of i5 (purple, 5001-5006) and i7 indices (yellow, 7001 – 7006). Using combinatorial dual indexing the same i5 and i7 indices can be used for different libraries, so long as the other index is unique (e.g., libraries B and C both have i5 index 5002, but different i7 indices (7002 or 7003)). For truly unique dual indexing, each i5 and i7 index can be used only once, for a single library. In this way only a maximum of 96 uniquely dual-indexed libraries can be multiplexed in a single sequencing lane or run.

Combinatorial dual indexing greatly enhances the multiplexing capacity of a sequencing lane or run. Using Lexogen’s 96 i5 x 96 i7 indices in combination, up to 9,216 libraries can be uniquely barcoded for multiplexed sequencing in a single run or lane. In doing so, the same i7 and i5 indices can be used for different individual libraries in the pool, so long as the i5 / i7 combination is unique for each library.

Unique Dual Indexing differs from combinatorial dual indexing, whereby each i5 and i7 index is present only once in the pool of multiplexed libraries. Unique dual indexing is required to detect index hopping and prevent read mis-assignment during demultiplexing. Index hopping can happen when either the i7 or i5 index of a particular library fragment is switched during cluster generation. Index hopping occurs more frequently on patterned flow cell instruments (HiSeq 3000 / 4000 and NovaSeq) that use ExAmp chemistry, and when excess indexing primers, or adapter dimers are present.

Index hopping can also be controlled by repurifying library pools prior to sequencing to remove excess adapter dimers and free index primers. Lexogen’s Purification Module with Magnetic Beads (Cat. No. 022.96) can be used for this repurification step. Briefly, add 0.9 volumes of Purification beads (PB) to the library pool, mix well and incubate for 5 minutes at room temperature, then follow steps 30 onwards in the QuantSeq User Guide Detailed protocol (see 015UG009 for protocol details).

Hamming distance refers to the number of nucleotide exchanges needed to convert one index sequence to another. All Lexogen i5 and i7 indices are designed with a minimum hamming distance of 3, meaning that each i7 index differs from all other i7 indices by at least 3 nucleotides, and each i5 index differs from all other i5 indices by at least 3 nucleotides. For example, considering 7001: CAGCGT and 7003: ACCAGT, the underlined nucleotides 1 – 4 would all need to be changed or mismatched to convert 7001 to 7003, or vice versa. Therefore, the hamming distance between these index sequences is 4.
With a hamming distance of 3, you can detect up to 2 errors (mismatches). However, if you want to perform error correction, only 1 error (or mismatch) can be detected and corrected. Turning off error correction during demultiplexing and allowing for zero mismatches, can result in higher rates of unidentified reads. However, this will increase the accuracy of index identification giving you more accurate sequencing reads for each library. Using (unique) dual indexing, can further enhance read identification accuracy as it prevents read mis-assignment (see FAQ 4.6).

Downloads

pdf  QuantSeq Application Note (Nature Methods, December 2014) – external link
pdf  Application Note for QuantSeq
pdf  Product Flyer – introducing QuantSeq data analysis in Partek Flow
pdf  Application Note for Globin Block Modules
pdf  Application Note for Automation of QuantSeq 3’ mRNA Kit on the Agilent NGS Workstation
pdf  Application Overview Flyer for Automation of QuantSeq 3’ mRNA Kit on Beckman Biomek NGS Workstation

QuantSeq 3′ mRNA-Seq Library Prep Kit REV for Illumina

pdf  User Guide – update 26.02.2019
pdf  User Guide – QuantSeq 3’ mRNA-Seq Integrated Data Analysis Pipelines on Bluebee® Genomics Platform

pdf
Globin Block Modules Instruction Manual – release 19.12.2017
pdf PCR Add-on Kit for Illumina Instruction Manual – update 03.07.2019
pdf Lexogen i5 6 nt Dual Indexing Add-on Kits (5001-5096) Instruction Manual – update 12.03.2019

pdf Lexogen i7 and i5 Index Sequences – including for kits bought before 17.02.2017
pdf Library Quantification Calculation File

autoQuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina

      • Perkin Elmer: Sciclone® / Zephyr®
      • Hamilton: Microlab STAR / STARlet
      • Agilent: NGS Workstation (NGS Bravo Option B)
      • Beckman Coulter: Biomek FXP, and Biomek i7
      • Eppendorf: EpMotion® 5075

Please inquire at info@lexogen.com for the automation scripts

Material Safety Datasheets

pdf  MSDS information for QuantSeq Expression Profiling Library Prep Kits – update 17.02.2016 (protocol adjusted to the upgraded QuantSeq protocol)
pdf  MSDS information for Globin Block Modules for QuantSeq – release 11.04.2018

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

QuantSeq Bioinformatics Data Analysis

Find more about the QuantSeq Data Analysis here.

QuantSeq 3’ mRNA-Seq Free Trial Kit

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