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
Mapping of Transcript End Sites
Direct Counting for Gene Expression Quantification
Just one fragment per transcript is produced; therefore, no length normalization is required. This allows more accurate determination of gene expression values and renders QuantSeq the best alternative to microarrays and conventional RNA-Seq in gene expression 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.
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.
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).
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. Please contact Lexogen at support@lexogen.com for input on determining the optimal endpoint cycle number.
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.
- First Strand cDNA Synthesis
-
- At step 3 pre-warm the FS2 / E1 mastermix for 2 – 3 minutes at 42 °C while the RNA / FS1 samples are denaturing for 3 minutes at 85 °C – Do not cool the mastermix on ice!
- After the RNA / FS1 samples have cooled to 42 °C, spin these down briefly and then immediately return to the thermocycler and hold at 42 °C.
- Add the pre-warmed FS2 / E1 mastermix to the RNA / FS1 samples on the thermocycler at 42 °C (step 4) and mix properly. Any drop in temperature at this point can cause mishybridization! Seal the plate or tubes and begin the 42 °C incubation.
NOTE! Spin down the samples at room temperature before and after adding the FS2 / E1 mastermix.
-
- If step is skipped (low input or degraded samples i.e. ≤10 ng, or FFPE samples):
- Prepare your RNA samples in 5 ul volumes
- 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.
- Bring your RNA samples to room temperature while the mastermix is pre-warming.
- Spin down the pre-warmed FS1 / FS2 / E1 mastermix and add 15 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:
- After the 42 °C incubation is complete spin down the plate/tubes briefly and place at room temperature.
- Immediately add the RNA Removal Solution (RS, thawed at room temperature) to the samples, mix well.
- Briefly spin down the plate / tubes at room temperature, then place on the thermocycler to commence the 10 minute incubation at 95 °C (step 6).
- 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:
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.
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.
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.
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.
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.
- 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.
Downloads
QuantSeq Application Note (Nature Methods, December 2014) – external link
Application Note
QuantSeq 3′ mRNA-Seq Library Prep Kit for Ion Torrent
User Guide – update 13.06.2018
Send us your publication & get the RNA T-shirt!
QuantSeq for Ion Torrent In-line Barcodes Overview
Material Safety Datasheets
MSDS Information can be found in the Documents page.
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