Description

QuantSeq 3’ mRNA-Seq Library Prep Kits

The QuantSeq Kit is a library preparation protocol designed to generate Illumina and Ion Torrent compatible libraries of sequences close to the 3’ end of the polyadenylated RNA.

QuantSeq kits are available in two versions. QuantSeq FWD generates NGS reads towards the poly(A) tail, directly reflecting the mRNA sequence. It is offered for both Illumina and Ion Torrent platforms. QuantSeq Reverse (REV) is only available for Illumina platforms and requires a Custom Sequencing Primer (CSP Version 2, included in the kit) for Read 1. With QuantSeq REV and the CSP it is possible to exactly pinpoint the 3’ end in Read 1 as 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 Quality Samples

The required input amount of total RNA is as low as 100 pg (QuantSeq for Illumina) and 5 ng (QuantSeq for Ion Torrent). 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 any user even without bioinformatics background. Access to the pipeline is free of charge for any QuantSeq 3’ mRNA-Seq for Illumina customer. 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 TES

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 external i7 indices (BC01-96) 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 detailed information about indices (barcodes) and instructions how to use them please consult Appendix D: Multiplexing, QuantSeq for Ion Torrent User Guide (page 23) and Appendix E: Multiplexing, QuantSeq for Illumina User Guide (page 25) and the i5 Dual Indexing Add-on Kit for QuantSeq/SENSE Instruction Manual.

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 sequencing
platform-compatible linker sequences.
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 also contains sequencing
platform-compatible linker sequences.
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 the complete sequences required
for cluster generation on Illumina platforms or colony formation on
Ion Torrent platforms are introduced.
Library Amplification
Step 4:
Multiplexing can be performed with up to 96 external barcodes for Illumina
and 48 in-line barcodes for Ion Torrent sequencing platforms.

For viewing the whole workflow on page please click here

Featured Publications

List of the most recent QuantSeq 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 96 Illumina-compatible libraries of the sequences close to the 3’ end of the polyadenylated RNA within 7 – 8.5 hours depending on the liquid handler used.

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 and the Hamilton Microlab STAR Workstations. 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 7 hours (on the PerkinElmer Sciclone/Zephyr) or 8.5 hours (on the Hamilton STAR), 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 autoQuantSeq kit is appropriate for preparing 96 barcoded libraries. The program on the PerkinElmer Sciclone/Zephyr liquid handler allows for processing of samples in multiples of 8 reactions (full columns of a 96-well plate), while on the Hamilton STAR any number of reaction from 1- 48 can be run. The reagents from a single kit can be consumed over several machine runs.

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).

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, 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 353 – 384 bp, with mean insert sizes of 231 – 262 bp. To generate longer libraries, use the QuantSeq-Flex First Strand Synthesis Module (Cat. No. 026).
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, mouse tissues, 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) SS1 in step 8 PS used in step 17 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 std SS1 72 μl 122 1500 384 262 99 % 81 % 28 % 2.5 12.3 11
500 ng std SS1 72 μl 122 1500 381 259 96 % 81 % 33 % 2.4 11.4 12
50 ng std SS1 72 μl 122 1500 347 225 98 % 78 % 27 % 1.9 9.9 15
10 ng std SS1 48 μl 122 1000 405 283 97 % 80 % 33 % 2.5  12.4 18
5 ng std SS1 48 μl 122 1000 447 325 97 % 86 % 42 % 2.1 12.1 20
500 pg std SS1 48 μl 122 1000 353 231 97 % 75 % 28 % 1.6 8.2 23

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

Lower inputs (10 ng or less) may require protocol adjustments, such as reducing the addition of PS in step 17 to 48 µl. Even so, an additional purification of the lane mix with 1 x PB (e.g., 50 µl lane mix plus 50 µl PB) and following the protocol from step 29 on again may be necessary, especially for less than 500 pg total RNA input to prevent sequencing through poly(A) stretches and remove all library fragments below 150 bp (inserts smaller than 38 bp). For more information regarding the input RNA requirements please consult Appendix B (p. 21). Please note that for QuantSeq REV we do not recommend to use input amounts lower than 10 ng total RNA.

QuantSeq FWD was successfully tested with as little as 10 pg of Universal Human Reference (UHR) RNA input. When using less than 1 ng of total RNA input please follow these recommendations.

    1. Skip step 2, immediately proceed to step 3.
    3. Extend the time of the RT in step 4 to 1 h.
    4. Reduce the time in step 6 to 5 min at 95 °C.
    5. Use 48 µl PS in step 17.
    6. Use 27 µl PB in step 30.
    7. Perform qPCR to determine the exact number of cycles for the endpoint PCR

For QuantSeq REV we recommend not to use input amounts lower than 10 ng total RNA.

The number of cycles for your endpoint PCR depends on the type of the RNA (tissue, organism), the RIN number and the RNA input amount. The reference values given in Appendix B, 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 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. In step 25 add another 17 µl of EB or H2O to your eluted libraries so 17 µl can be used for the qPCR and 17 µl for the subsequent endpoint PCR. Follow steps 26-27, add 5 µl of Barcode 00 (BC00) and add SYBR Green I (diluted in DMSO) to a final concentration of 0.1x and 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 33% of the maximum (see Fig. 3). The value where the fluorescence reaches the maximum (plateau) is taken (13744) and the fluorescence at 33% of this values (4535) shows which cycle number is optimal for the endpoint PCRs. For the sample in Fig. 3 this would be 11 cycles, but also 12 cycles can be used if a higher yield is preferred.Calculation_of_the_number_of_cycle_for_the_endpoint_PCR

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

This cycle number is to be used for the endpoint PCR with the remaining half of your sample. Once the number of cycles for the endpoint PCR is established for one type of sample, you can use it in the following experiments and reduce the number of cycle by one as cDNA dilution for an additional qPCR is no longer required.

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

Input RNA (500 ng) Cycles ng/µl nM
UHRR 12 2.4 11.2
HBRR 13 4.0 20.8
Hs. ES 10 1.2 6.6
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. ES 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 3.4 19.6
Tomato seeds 16 1.7 9.4
Fungi RNA 13 1.24 7.08
Yeast RNA (S.c.) 12 1.2 7.7

Yes, low quality and FFPE samples can be used with QuantSeq. Some minor protocol modification are required though:

  1. Skip step 2, immediately proceed to step 3.
  2. Use 48 µl PS in step 17,
  3. use 27 µl in step 30.

Please be aware that more PCR cycles are necessary (see Table).

ng FFPE RNA Input Recommended
cycle number
50 ng FFPE 18
10 ng FFPE 24
500 pg FFPE 27
Lexogen’s QuantSeq kit is a library preparation protocol designed to generate up to 96 sequence-ready Illumina-compatible libraries from polyadenylated RNA within 4.5 hours. When you carry 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 (BC01-96) 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.
QuantSeq FWD libraries can be easily multiplexed with samples from other library preps. However, QuantSeq REV cannot be multiplexed with other library preps as the Custom Sequencing Primer is needed for sequencing. QuantSeq barcodes differ from Illumina’s. Exception is a barcode 5 (TAATCG) which is identical to the Illumina barcode 42.
QuantSeq FWD (Cat. No. 015) generates NGS reads towards the poly(A) tail. To pinpoint the exact 3’ end, longer read lengths may be required. Read 1 directly reflects the mRNA sequence.
With QuantSeq REV and the custom sequencing primer it is possible to exactly pinpoint the 3’ end during Read 1. The reads generated here during Read 1 reflect the cDNA sequence, so they are in a strand orientation opposite to the genomic reference.
STAR aligner or TopHat2 can be used for mapping QuantSeq FWD (Cat.No. 015) data. The reads may not land in the last exon and span a junction. For QuantSeq REV (Cat.No. 016) we do not recommend using TopHat2, since there is hardly a 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.
As second strand synthesis is based on random priming, there may be a higher proportion of errors at the first nucleotides of the insert due to non-specific hybridization of the random primer to the cDNA template. These mismatches can lead to a lower percentage of mappable reads when using a stringent aligner such as TopHat2 in which case it may be beneficial to trim these nucleotides. For QuantSeq FWD (Cat. No. 015) the first twelve nucleotides need to be removed from Read 1. Alternatively, a less stringent aligner (e.g., STAR Aligner) could be used with the number of allowed mismatches being set to 14. While trimming the first nucleotides can decrease the number of reads of suitable length, the absolute number of mapping reads may increase due to the improved read quality. Reads which are too short or have generally low quality scores should be removed from the set.
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, also here the 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 kit (FWD, REV) are appropriate for HiSeq 2000/2500, HiSeq 3000, HiSeq 4000, GAIIX, MiSeq, while on NextSeq 500 and NextSeq 550 Illumina platforms only QuantSeq FWD can be used.
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 >SE 50 available for mapping in order to increase the number of uniquely mapping reads.
For paired-end sequencing we generally recommend using QuantSeq REV. Using QuantSeq FWD the quality of Read 2 would be very low due to the poly(T) stretch at the beginning of Read 2. For the library preparation itself we recommend using only 24 µl of PB in step 30 of the QuantSeq protocol (Purification, QuantSeq User Guide, page 15). In this way, inserts smaller than 50 bp are more efficiently removed and the quality of the sequencing run will improve as the likelihood of reading into poly(T) stretches decreases. For step 31-39 simply follow the User Guide.
For QuantSeq REV it is essential to use the CSP 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 PDF 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 (RT) between 20 °C and 25 °C. To prevent mis-priming during reverse transcription 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).
  • Addition of the RS1 and RS2 solutions, they have to be added in an equal amount, otherwise you will get differences in the yield.
  • RS2 and SS1 have to be added in sequential order. Never mix RS2 and SS1 directly with each other as this will negatively affect the library prep.
  • During the magnetic bead-based purification take care to not dry the beads too long (visual cracks will appear) as this will negatively influence the elution, but also don´t carry over traces of EtOH to the next reactions.
  • Perform all steps at room temperature (including centrifugation) and don´t put your samples on a cooling block or on ice.
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 38 (Purification, QuantSeq User Guide, page 15). Leave approximately 2 µl of the eluate on the beads if a complete removal of the supernatant without beads carryover is not possible.
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. In general, however, 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 lanemix were undercycled.
Universal Human Reference RNA (UHRR, Agilent) is a good positive control, the 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.
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
Various multiplexing options are available to suit your experimental design. However, care should be taken to always use sets of barcodes that give a signal in both color channels for each nucleotide position. In detail, at least one of the two bases A or C (red channel) AND one of the two bases G or T (green channel) should be present at a given nucleotide position for all Illumina sequencers except NextSeq machines which uses a different color coding system.

In general we recommend using a complete set of 8 or 12 barcodes for multiplexing (e.g., Set 1 or Set A if the 96 reaction kit is used, respectively). However, if fewer barcodes are required also subsets of each set can be chosen.

Two samples per lane: In step use 2.5 µl of BC01 and 2.5 µl BC13 for one sample and 2.5 µl BC25 and 2.5 µl BC37 for the second. Here two barcodes are applied to each sample in order to balance the red and green laser signals.
Four samples per lane: In step use 5 µl of BC01 for one sample, 5 µl BC13 for the second, 5 µl BC25 for the third, and 5 µl BC37 for the fourth. Apply only one barcode to each sample.

Eight samples per lane: In step use all the barcodes from Set 1 (BC01/BC13/BC25/BC37/ BC49/BC61/BC73, and BC85). Apply only one barcode to each sample.

Twelve samples per lane: In step use all the barcodes from Set 1 (BC01/BC13/BC25/ BC37/BC49/BC61/BC73, and BC85) plus 4 barcodes from Set 2 (BC02/BC14/BC26, and BC38) if you have the 24 reaction kit (Cat. No. 001.24). Alternatively, if using the 96 reaction kit (Cat. No. 001.96) barcodes BC01 – 12 from row A can be used. Apply only one barcode to each sample.

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The QuantSeq protocol is optimized for shorter reads (SR100) and yields mean insert sizes of about 200 to 250 bp.
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, mouse tissues, 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 5 ng without adjusting the protocol. However, for most efficient detection of low abundant transcripts RNA inputs from 500 ng – 200 ng are recommended.

Input RNA (UHR) PS used in step 17 Library* Insert Library yield PCR cycles
 Start [bp]  End [bp] Mean size* Mean size ≥ 50 nt ≥ 100 nt ≥ 200 nt ≥ 300 nt ≥ 400 nt  ng/μl  nM
500 ng 56 μl 100 1500 331 260 98 % 76 % 29 % 11 % 4 % 2.2 13.0 11
50 ng 56 μl 100 1500 298 227 97 % 71 % 24 % 8 % 2 % 1.6 10.0 14
10 ng 56 μl 100 1500 290 219 94 % 70 % 23 % 6 % 2 % 1.8  11.4 17
5 ng 56 μl 100 1500 294 223 94 % 70 % 24 % 7 % 2 % 1.2 7.9 17

*All libraries are prepared with in-line barcode(BC01). Linker sequences are 84 bp including the 9 nt long in-line barcodes.

When using less than 5 ng total RNA input, some protocol adjustments, such as increased number of PCR cycles and reduction of PS addition in step 17 (48 µl PS) are needed. A further purification of the lane mix would be advisable in order to remove all library fragments below 125 bp (inserts smaller than 41 bp). For more information regarding the input RNA requirements please consult Appendix B (p. 20).

Yes, low quality and FFPE samples can be used with QuantSeq, but some minor protocol modifications are necessary. Please consult support@lexogen.com for further information.
Lexogen’s QuantSeq kit is a library preparation protocol designed to generate up to 48 sequence-ready Ion Torrent-compatible libraries from polyadenylated RNA within 4.5 hours. When you carry 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 in-line barcodes during second strand synthesis, allowing up to 48 samples to be sequenced per lane. In-line barcodes are 9 – 11 nt long followed by an additional 4 nt constant sequence (CGAT) and compose the first 17 – 19 nt nucleotides of the read (TCAG – barcode – CGAT).
QuantSeq barcodes 01-24 and IonXpress barcodes 01-24 are of identical sequence, so in fact you can simply select IonXpress barcodes on your machine. The only difference is that we consider the C present in all barcodes a part of the constant region whereas IonTorrent counts the constant C as the last nucleotide of the barcode and only the GAT as constant part. When multiplexing QuantSeq libraries with IonXpress barcoded libraries make sure to use different barcodes e.g., QuantSeq barcodes 1-48 with IonXpress barcodes 49-96.
Barcode Set A contains in-line barcodes 01-24 while in Barcode Set B in-line barcodes 25-48 are include. See also consult Appendix D: Multiplexing, QuantSeq for Ion Torrent User Guide (page 23).
The reads are directly reflecting the mRNA sequence and no re-orientation is necessary.

STAR aligner or bbmap can be used for mapping. As QuantSeq is a 3’ Seq protocol, most sequences will originate from the last exon and the 3’ untranslated region (UTR).

Alternatively TMAP mapping program can be used, as this program is optimized for aligning reads of variable length. It includes three algorithms that may be run together (mapall) or individually (map1, map2, and map3). For RNA-Seq seed lengths of 18 nucleotides and employing the default number of allowable mismatches per seed are commonly used.

As second strand synthesis is based on random priming, there may be a higher proportion of errors at the first nucleotides of the insert due to non-specific hybridization of the random primer to the cDNA template. These mismatches can lead to a lower percentage of mappable reads when using a stringent aligner in which case it may be beneficial to trim the first 12 nucleotides. Alternatively a less stringent aligner (e.g., STAR Aligner) could be used with an increased number of allowed mismatches.
While trimming the first nucleotides can decrease the number of reads of suitable length, the absolute number of mapping reads may increase due to the improved read quality. Reads which are too short or have generally low quality scores should be removed from the set.
For trimming we recommend using the FASTX-toolkit available from the Hannon lab (CSHL) or the trimming functions of the bbmap suite bbmap suite (although the functionality of the mapper on QuantSeq reads has not yet been fully evaluated).
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 insert size is optimized for shorter reads (SR100). However, longer read lengths are also possible if a more detailed analysis of the very 3’ end of transcripts is desired. Read lengths longer than SR100 can be chosen (e.g., SR200, SR300, SR400), if the exact 3’ end is to be pinpointed for most transcripts. Be aware that adapter reads will increase with longer reads length. In this case trimming the adapter sequences before mapping is essential.
A second peak between 100 – 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, 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 38 (Purification, QuantSeq User Guide, page 15). Leave approximately 2 µl of the eluate on the beads if a complete removal of the supernatant without beads carryover is not possible.
QuantSeq works fine with the HiQ system, however there seems to be a problem with the IonChef. Switch back to the OneTouch System 2 to get the great results with the HiQ system.
The QuantSeq 3’ mRNA-Seq kit (012.24) is appropriate for the Ion Proton and Ion PGM Systems.
  • Proper mixing of the viscous solutions (such as BC01-48, 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).
  • Addition of the RS1 and RS2 solutions, they have to be added in an equal amount, otherwise you will get differences in the yield.
  • RS2 and SS1 have to be added in sequential order. Never mix RS2 and SS1 directly with each other as this will negatively affect the library prep.
  • During the magnetic bead-based purification take care to not dry the beads too long (visual cracks will appear) as this will negatively influence the elution, but also don´t carry over traces of EtOH to the next reactions.
  • Perform all steps at room temperature (including centrifugation) and don´t put your samples on a cooling block or on ice.
Universal Human Reference RNA (UHRR, Agilent) is a good positive control, the most of the reference values given in the User Guide are also based on UHRR input.

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The autoQuantSeq mRNA-Seq protocol is currently automated on PerkinElmer Sciclone NGS workstation, using the Zephyr machine for the post-PCR purification and on the Hamilton Micolab STAR liquid handlers. 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.

For the PerkinElmer Sciclone any number of columns from 1 to 12 (simply by changing the respective entry in the Excel Workbook) can be run. The machine will always take the barcodes from the first N wells of the P6 plate, starting with column 1. Therefore, you can run an assay with less than 96 (for example 24) reactions in multiples of 8, but you have to prepare a new P6 plate which will substitute the barcode plate contained in the kit. You can do this by simply transferring the reagents from the kit barcode plate to the columns 1, 2, and 3 (24 reactions = 3 columns x 8 rows) of a new HSP96 plate.

The procedure is also described in detail in the autoQuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina on PE Sciclone/Zephyr User Guide (page 21).

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 only an off-deck option is available.
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. 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.

Downloads

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

QuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina

pdf  User Guide – update 01.12.2016 (Use of QuantSeq-Flex First Strand Synthesis Module to generate longer libraries; Lowest recommended input for QuantSeq REV 10 ng; Thawing of SS1 buffer at 37 °C. Information about the integrated QuantSeq 3’mRNA-Seq Data Analysis Pipeline on the Bluebee Genomics Platform)
pdf
PCR Add-on Kit for Illumina Instruction Manual
pdf i5 Dual Indexing Add-on Kit for QuantSeq/Sense Instruction Manual

pdf
  Product Flyer
pdf  QuantSeq for Illumina External Indices (Barcodes) Overview

QuantSeq 3′ mRNA-Seq Library Prep Kit for Ion Torrent

pdf  User Guide – update 15.03.2016 (Temperature of reverse transcription raised from 37 °C to 42 °C to get less unspecific hybridization; Protocol adjustments for or low input, FFPE, or low quality RNA in step 2, 6, 17, and 29.)
pdf PCR Add-on Kit for Ion Torrent Instruction Manual
pdf  QuantSeq for Ion Torrent In-line Barcodes Overview

autoQuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina

pdf  User Guide – update 15.12.2015 (Updates to match QuantSeq User Guide; Temperature of reverse transcription raised from 37 °C to 42 °C to get less unspecific hybridization; Off deck thermal treatment for reverse transcription introduced.)
zip  autoQuantSeq Ver.2016-01-08a (FULL PACKAGE)
pdf  autoQuantSeq Ver.2015-12-18a (WORKBOOK ONLY)
pdf  User Guide – update 16.12.2015 (Temperature of reverse transcription raised from 37 °C to 42 °C to get less unspecific hybridization; Additional manual intervention and thermocycler program added; Updated handling recommendation.)
zip  autoQuantSeq Hamilton Ver.2015-12-18a (FULL PACKAGE)
pdf  autoQuantSeq Hamilton Ver.2015-12-18a (WORKBOOK ONLY)

Material Safety Datasheets

pdf  MSDS information for QuantSeq Expression Profiling Library Prep Kits

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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