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
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/. For using your activation code register an account with BlueBee (https://lexogen.bluebee.com/portal) and upload your data (fastq.gz files).
NOTE! BlueBee Data Analysis is available for a range of species. Reference genomes for new species can be added upon request. Please note this will incur a fee. See QuantSeq Data Analysis, for further details.
Mapping of Transcript End Sites
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.
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.
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.
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.
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.
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. |

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.
ATTENTION: QuantSeq REV cannot be multiplexed with other library preps as the Custom Sequencing Primer is needed for sequencing.
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
- First Strand cDNA Synthesis (p.11-12):
- 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!
- 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.
- 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):
- Prepare your RNA samples in 5 μl volumes.
- 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.
- Bring your RNA samples to room temperature while the mastermix is pre-warming.
- 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:
- After the 42 °C incubation is complete spin down the plate/tubes briefly and place at room temperature.
- Immediately add the RNA Removal Solution (RS, thawed at room temperature) to the samples, mix well.
- 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.
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.
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.
The code supplied with the QuantSeq REV Kits (Cat. No. 015) enables only the REV data analysis pipeline to be selected. Do not use Activation codes from QuantSeq FWD kits for analysis of QuantSeq REV data!
Each code contains an equal number of pipeline runs as reactions provided in the kits (e.g. You get 96 data analysis runs with a 96 prep QuantSeq REV Kit (Cat. No. 016.96), meaning you can analyze sequencing data for 96 .fastq files).
If you start the wrong pipeline for your data you can abort or stop the run before it is completed without losing your allocated runs. If the run is completed then additional runs will need to be purchased (Cat. No. 091).
QuantSeq FWD libraries prepared from blood using Globin Block (RS-GBHs or RS-GBSs, Cat. No. 070 and 071) should be analysed using the standard REV pipeline for data analysis.
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 | Arabidopsis halleri |
Arabidopsis Thale cress | Arabidopsis thaliana |
Baker’s Yeast | Saccharomyces cerevisiae |
Barley | Hordeum vulgare |
Bartonella | Bartonella henselae |
Brain-Eating Amoeba | Naegleria fowleri |
Chicken | Gallus gallus |
CHO-K1 Cell Line | Cricetulus griseus |
Common Yellow Monkeyflower | Mimulus guttatus |
COVID19 | SARS-CoV-2 |
Cow | Bos taurus |
Cacao Tree | Theobroma cacao criollo |
Dog | Canis lupus familiaris |
Drummond’s Rockcress | Boechera stricta |
Ferret | Mustela putorius furo |
Fruit Fly | Drosophila melanogaster |
Fungus | Fusarium oxysporum |
Fungus | Yarrowia lipolytica |
Goat | Capra hircus |
Human | Homo sapiens |
Maize/Corn | Zea mays |
Melon Fly | Bactrocera cucurbitae |
Mouse | Mus musculus |
Nematode Roundworm | Caenorhabditis elegans |
Painted Turtle | Chrysemys picta bellii |
Pig | Sus scrofa |
Potato | Solanum tuberosum |
Pseudomonas | Pseudomonas aeruginosa PA103 |
Purple False Brome | Brachypodium distachyon |
Rabbit | Oryctolagus cuniculus |
Rat | Rattus norvegicus |
Rice | Oryza sativa |
Salmon | Salmon salar |
Sorghum | Sorghum bicolor |
Sponge | Amphimedon queenslandica |
Starlet Sea Anemone | Nematostella vectensis |
Tomato | Solanum lycopersicum |
Water flea | Daphnia pulex |
Western Balsam Poplar Tree | Populus trichocarpa |
Yeast | Candida albicans, Candida auris, Candida parapsilosis |
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.
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.
Figure 4 | Location of the BlueBee® Data Analysis activation code on QuantSeq Kit reagent boxes.
For further information about the i5 Dual Indexing Add-on Kit for QuantSeq/SENSE please see the QuantSeq REV online FAQs 4.1 onwards.
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).
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- 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.
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.
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.
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.
Globin Block libraries can also display: a longer library size distribution, and/or, peaks present at larger molecular weights (~400 bp and higher).

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).
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 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.
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- 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)
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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.
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- 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).
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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.
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).
There are two types of dual indexing: combinatorial dual indexing, and unique dual indexing (Fig. 1).
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).
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
QuantSeq Application Note (Nature Methods, December 2014) – external link
Application Note for QuantSeq
Product Flyer – introducing QuantSeq data analysis in Partek Flow
Application Note for Globin Block Modules
Application Note for Automation of QuantSeq 3’ mRNA Kit on the Agilent NGS Workstation
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
User Guide – update 03.04.2020
Send us your publication & get the RNA T-shirt!
User Guide – QuantSeq 3’ mRNA-Seq Integrated Data Analysis Pipelines on BlueBee® Genomics Platform – update 20.01.2021
PCR Add-on Kit for Illumina Instruction Manual – update 04.11.2020
Lexogen i5 6 nt Dual Indexing Add-on Kits (5001-5096) Instruction Manual – update 12.03.2019
Lexogen i7 and i5 Index Sequences – update 05.05.2020
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
MSDS Information can be found in the Documents page.
If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.
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Find more about the QuantSeq Data Analysis here.
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