Description

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

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

The QuantSeq protocol is designed to yield sequences close to the 3’ end of polyadenylated RNAs, whereat only one fragment per transcript is produced. With QuantSeq for Ion Torrent NGS reads are generated towards the poly(A) tail. Reads directly reflect the mRNA sequence.

Analysis of Low Quality Samples

The required input amount of total RNA is as low as 5 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.

Cost Saving Multiplexing

QuantSeq libraries are intended for a high degree of multiplexing. 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 (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 27).

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

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

Direct Counting for Gene Expression Quantification

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

Workflow

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

Reverse Transcription
Step 1:
The kit uses total RNA as input, hence no prior poly(A) enrichment
or rRNA depletion is needed.
01quantseq_workflowtime_quantseq01
Reverse Transcription
Step 1:
Library generation starts with oligodT priming containing the
Ion Torrent-specific P1 adapter in the 5`part of the oligodT primer.
02quantseq_workflowtime_quantseq02
Removal of RNA
Step 2:
After first strand synthesis the RNA is removed.
03quantseq_workflowtime_quantseq03
Second Strand Synthesis
Step 3:
Second strand synthesis is initiated by random priming and a DNA
polymerase. The random primer contains the Ion Torrent-specific
A adapter in the 5’ end of the random primer and up to 48 in-line
barcodes can be introduced.
04quantseq_workflow_ittime_quantseq04
Second Strand Synthesis
Step 3:
No purification is required between first and second strand synthesis.
Second strand synthesis is followed by a magnetic bead-based
purification step rendering the protocol compatible with automation.
05quantseq_workflow_ittime_quantseq05
Library Amplification
Step 4:
During the library amplification step the sequences required
for colony formation are introduced.
06quantseq_workflow_ittime_quantseq06

For viewing the whole workflow on page please click here

Multiplexing

In QuantSeq for Ion Torrent up to 48 barcodes (Barcode Set A and B) are introduced as standard in-line barcodes during the second strand synthesis step.

Barcode Set A Barcode Set B
1 2 3 4 5 6
 A BC01:
CTAAGGTAA
BC09:
TGAGCGGAA
BC17:
TCTATTCGT
BC25:
CCTGAGATA
BC33:
TTCTCATTGAA
BC41:
TTCCACTTCG
 B BC02:
TAAGGAGAA
BC10:
CTGACCGAA
BC18:
AGGCAATTG
BC26:
TTACAACCT
BC34:
TCGCATCGTT
BC42:
AGCACGAAT
C BC03:
AAGAGGATT
BC11:
TCCTCGAAT
BC19:
TTAGTCGGA
BC27:
AACCATCCG
BC35:
TAAGCCATTGT
BC43:
CTTGACACCG
 D BC04:
TACCAAGAT
BC12:
TAGGTGGTT
BC20:
CAGATCCAT
BC28:
ATCCGGAAT
BC36:
AAGGAATCGT
BC44:
TTGGAGGCCAG
 E BC05:
CAGAAGGAA
BC13:
TCTAACGGA
BC21:
TCGCAATTA
BC29:
TCGACCACT
BC37:
CTTGAGAATGT
BC45:
TGGAGCTTCCT
 F BC06:
CTGCAAGTT
BC14:
TTGGAGTGT
BC22:
TTCGAGACG
BC30:
CGAGGTTAT
BC38:
TGGAGGACGGA
BC46:
TCAGTCCGAA
 G BC07:
TTCGTGATT
BC15:
TCTAGAGGT
BC23:
TGCCACGAA
BC31:
TCCAAGCTG
BC39:
TAACAATCGG
BC47:
TAAGGCAACCA
 H BC08:
TTCCGATAA
BC16:
TCTGGATGA
BC24:
AACCTCATT
BC32:
TCTTACACA
BC40:
CTGACATAAT
BC48:
TTCTAAGAGA

Featured Publications

Automation

autoQuantSeq 3’ mRNA-Seq Library Prep Kit for Ion Torrent

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 48 Ion Torrent-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 is compatible with automation and Lexogen provides automated protocols and software for diverse platforms. If you are interested in an automated protocol or need help automating QuantSeq on your NGS workstation, please contact Lexogen.

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.

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. 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.

The minimum recommended input is 5 ng of high quality total RNA. When using low quality or degraded RNA, or FFPE RNA input, the recommended protocol modifications are outlined in the table below:

Protocol Step Standard Input (>5 ng) Low quality / degraded / FFPE Input
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 17 Add 56 μl of Purification Solution (PS). Reduce volume of Purification Solution (PS) to 48 μl.
Step 25 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 36 μl of Purification Beads (PB). Reduce volume of Purification Beads (PB) to 30 μl.

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. 23).

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 (>5 ng) Low quality / degraded / FFPE Input
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 17 Add 56 μl of Purification Solution (PS). Reduce volume of Purification Solution (PS) to 48 μl.
Step 25 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 36 μl of Purification Beads (PB). Reduce volume of Purification Beads (PB) to 30 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
500 pg FFPE 22

* 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.

  • First Strand cDNA Synthesis
      1. At step 3 pre-warm the FS2 / E1 mastermix for 2 – 3 minutes at 42 °C while the RNA / FS1 samples are denaturing for 3 minutes at 85 °C – Do not cool the mastermix on ice!
      2. After the RNA / FS1 samples have cooled to 42 °C, spin these down briefly and then immediately return to the thermocycler and hold at 42 °C.
      3. Add the pre-warmed FS2 / E1 mastermix to the RNA / FS1 samples on the thermocycler at 42 °C (step 4) and mix properly. Any drop in temperature at this point can cause mishybridization! Seal the plate or tubes and begin the 42 °C incubation.

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

  • If step is skipped (low input or degraded samples i.e. ≤10 ng, or FFPE samples):
    1. Prepare your RNA samples in 5 ul volumes
    2. Prepare a mastermix containing 5 ul FS1, 9.5 ul FS2, and 0.5 ul E1, mix well, spin down, and pre-warm at 42 °C on a thermocycler for 2 – 3 minutes.
    3. Bring your RNA samples to room temperature while the mastermix is pre-warming.
    4. Spin down the pre-warmed FS1 / FS2 / E1 mastermix and add 15 ul 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)).
    • Proceed immediately to the RNA removal after the reverse transcription is complete! Do not place the samples on ice, and do not store samples at -20 °C at this point! Cooling the samples below room temperature at this point can cause mishybridisation! Best practice handling would be as follows:
      1. After the 42 °C incubation is complete spin down the plate/tubes briefly and place at room temperature.
      2. Immediately add the RNA Removal Solution (RS, thawed at room temperature) to the samples, mix well.
      3. Briefly spin down the plate / tubes at room temperature, then place on the thermocycler to commence the 10 minute incubation at 95 °C (step 6).

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

  • When re-starting the protocol after safe stopping points where libraries are stored at -20 degrees, ensure that that they are thawed to room temperature before beginning the protocol again. This is particularly important for purification steps as reduced temperatures here can affect bead migration and library precipitation, leading to a loss of yield.
Lexogen’s QuantSeq kit is a library preparation protocol designed to generate ready-to-sequence 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 27).
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 41 (Purification, QuantSeq User Guide, page 16). 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.
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.

Downloads

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

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

pdf  User Guide – update 13.06.2018
pdf PCR Add-on Kit for Ion Torrent Instruction Manual
pdf  QuantSeq for Ion Torrent In-line Barcodes Overview

Material Safety Datasheets

pdf  MSDS information for QuantSeq Expression Profiling Library Prep Kits

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