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.

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/quantseq/

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.

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.

Perfect for Gene Counting

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 four external i5 indices (i5 Dual Indexing Add-on Kit, Cat. No. 047), up to 384 samples can be multiplexed and sequenced per lane on an Illumina flow cell.
In-line barcodes allowing up to 48 samples to be sequenced in one sequencing run of Ion Torrent instruments are included in QuantSeq 3’ mRNA-Seq Library Prep Kit for Ion Torrent. 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 H: Multiplexing, QuantSeq for Illumina User Guide (p. 28); Appendix A: Multiplexing, i5 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.
Reverse Transcription
Step 1:
Library generation starts with oligodT priming containing the
Illumina-specific Read 1 linker sequence.
Removal of RNA
Step 2:
After first strand synthesis the RNA is removed.
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.
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.
Library Amplification
Step 4:
During the library amplification step sequences required
for cluster generation are introduced.
Library Amplification
Step 4:
Multiplexing can be performed with up to 384 barcode combinations
using the 96 available i7 indices and four i5 indices.
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.

For viewing the whole workflow on page please click here

Featured Publications

List of the most recent QuantSeq publications.
List of the most recent webinars.

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 384 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 384 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.

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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 G, p.27.
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. If total RNA input ≤10 ng need to be used, please follow the recommendations below:

1. Skip step 2, immediately proceed to step 3
2. Use 48 µl PS in step 16.
3. Perform qPCR to determine the exact number of cycles for the endpoint PCR
4. Use 27 µl PB in step 29 for single indexing or 31.5 µl in step 30 for dual indexing PCR (or step 6 according to the i5 Dual Indexing Add-on Kit).

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). Conduct at least 30 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:

1. Skip step 2, immediately proceed to step 3.
2. Use 48 µl PS in step 16.
3. Perform qPCR to determine the exact number of cycles for the endpoint PCR
4. Use 27 µl PB in step 29 for single indexing or 31.5 µl in step 30 for dual indexing PCR (or step 6 according to the i5 Dual Indexing Add-on Kit).

ng FFPE RNA Input Recommended
Cycle Number
50 ng FFPE 15
10 ng FFPE 18

Please be aware the values in the table are based on Mm brain FFPE RNA with a RIN of 1.8 (DV200 of 51 %) and for different sources of RNA and RNA qualities more PCR cycles might be needed.

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 (i5 Dual Indexing Add-on Kit, Cat. No. 047), up to 384 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 of reverse transcription was too low. In particular, the centrifugation step in step 2 should not be carried out at 4 °C. Spin down at room temperature! As mentioned 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 and also mastermixes should not be cooled. To prevent mis-priming during reverse transcription you can leave the reaction at 42 °C and add the mastermix directly on the thermocycler, alternatively the reaction temperature can also be raised to 50 °C.
  • Proper mixing of the viscous solutions (such as SS1, PB, and PS) is really important. It can be facilitated when the buffers are at room temperature and if larger volumes are used for mixing (e.g., after adding 5 µl in steps 5 and 7, use a pipette set to 15 µl or 20 µl for mixing).
  • RS and SS1 have to be added in sequential order. Never mix RS and SS1 directly with each other as this will negatively affect the library prep.
  • During the magnetic bead-based purification make sure all the beads are collected at the magnet before taking the supernatant. Depending on the strength of your magnet incubation times need to be elongated. Take care to not dry the beads too long (visual cracks will appear) as this will negatively influence the elution, but also do not carry over traces of EtOH to the next reactions.
  • Perform all steps at room temperature (including centrifugation) and do not put your samples and mastermixes on a cooling block or on ice.
  • The optimal number of cycles is crucial for a sufficient yield. Performing a qPCR is recommended to determine the optimal number of cycles for the endpoint PCR in order to prevent any under- or overcycling.
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 E: qPCR and Reamplification, p.23). 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 values given in the table below are based on customer experiences. If you have already well established concentrations for your sequencer, you might want to try your standard concentration first.

Sequencer QuantSeq FWD QuantSeq REV
HiSeq 3000 / HiSeq 4000 / HiSeqXTen 280 pM
HiSeq 2000 / HiSeq 2500 10 pM 6.5 pM
MiSeq 6-15 pM 6-15 pM
NextSeq 500 / NextSeq 550 4.5 pM NOT RECOMMENDED
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.

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TThe autoQuantSeq 3′ mRNA-Seq protocol is currently automated on PerkinElmer Sciclone NGS workstation, using the Zephyr machine for the post-PCR purification, the Hamilton Micolab STAR liquid handlers, the Agilent Bravo Automated Liquid Handling Platform and the Biomek platforms of Beckman Coulter. The post-PCR can also be done manually or on any other liquid handler on which the PostPCR SPRI Purification application is established.

Lexogen is planning to automate the QuantSeq protocols on all other liquid handling platforms used for NGS. Please contact us if you are interested in putting QuantSeq on your platform.

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.

Downloads

pdf  QuantSeq Application Note (Nature Methods, December 2014) – external link
pdf  Application Note

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

pdf  User Guide – update 17.02.2017 (Upgrade of QuantSeq to a more streamlined protocol: a shortened RNA removal and adjusted second strand synthesis and purification steps; rearranged barcodes under i7 Index Plate)

pdf
PCR Add-on Kit for Illumina Instruction Manual – update 17.02.2017 (protocol adjusted to the upgraded QuantSeq protocol, BC00 was renamed to P7 Primer 7000)
pdf i5 Dual Indexing Add-on Kit for QuantSeq/SENSE Instruction Manual– update 17.02.2016 (protocol adjusted to the upgraded QuantSeq protocol)

pdf
  QuantSeq for Illumina Index Primer Overview (i7 Index Plate) – for kits bought after 17.02.2017
pdf Barcode Plate Overview – for kits bought before 17.02.2017

autoQuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina

  • PerkinElmrer Sciclone
  • Zephyr
Hamilton Mircrolab STAR
  • Agilent Bravo
  • Beckman Coulter Biomek

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)

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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Note: The trial kit is free of charge. However, please note that by requesting this trial kit, you agree to be charged for shipping expenses via invoice. The shipping costs for US customers are 35$ (US Dollars), and for the rest of the world - 39€ (Euro).

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