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.
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.
or rRNA depletion is needed.
Ion Torrent-specific P1 adapter in the 5`part of the oligodT primer.
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.
Second strand synthesis is followed by a magnetic bead-based
purification step rendering the protocol compatible with automation.
for colony formation are introduced.
For viewing the whole workflow on page please click here
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|
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.
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 email@example.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.
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).
Please stick to the recommendations given below.
1. Skip step 2, immediately proceed to step 3
2. Optional: Extend the time of the reverse transcription in step 4 to 1 h
3. Optional: Reduce the time in step 6 to 5 min at 95 °C
4. Use 48 µl PS in step 17
5. Use 30 µl PB in step 29
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.
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.
QuantSeq 3′ mRNA-Seq Library Prep Kit for Ion Torrent
User Guide – update 23.03.2017 (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.)
PCR Add-on Kit for Ion Torrent Instruction Manual
QuantSeq for Ion Torrent In-line Barcodes Overview
Material Safety Datasheets
If you need more information about our products, please contact us through firstname.lastname@example.org or directly under +43 1 345 1212-41.