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.

*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:

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:

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.

• First Strand cDNA Synthesis
  1. 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.