Description

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

SARS-CoV-2_Icon This product can be used for SARS-CoV-2 enquiry.

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

The QuantSeq protocol is designed to yield sequences close to the 3' cease 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 corporeality of total RNA is every bit low as v ng. QuantSeq is suitable to reproducibly generate libraries from depression 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.

Toll Saving Multiplexing

QuantSeq libraries are intended for a high caste 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 iii' 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 too designed to yield insert sizes for short sequencing reads (SR100).

For detailed information virtually indices (barcodes) and instructions how to apply them please consult Appendix D: Multiplexing, QuantSeq for Ion Torrent User Guide (page 27).

Rapid Turnaround

QuantSeq'due south simple workflow allows generating ready-to sequence-NGS libraries within only 4.v hours, including less than 2 hours hands-on fourth dimension.

Mapping of Transcript End Sites

Past using longer reads QuantSeq allows to exactly pinpoint the 3' cease of poly(A) RNA and therefore obtain accurate data about the three'UTR.

Directly Counting for Factor Expression Quantification

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

Workflow

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

For viewing the whole workflow on folio please click hither

Multiplexing

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

Barcode Ready 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
 Eastward 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
 Yard 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 upwards to 48 Ion Torrent-compatible libraries of the sequences close to the 3' finish of the polyadenylated RNA.

Automating the process of library preparation has the reward of avoiding sample tracking errors, dramatically increasing throughput, and saving easily-on time.

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

FAQ

Oftentimes Asked Questions

Please find a list of the most frequently asked questions beneath. If y'all cannot find the answer to your question here or want to know more about our products, please contact back up@lexogen.com.

The QuantSeq protocol is optimized for shorter reads (SR100) and yields mean insert sizes of most 200 to 250 bp.
The kit uses full 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 full 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 arable transcripts RNA inputs from 500 ng – 200 ng are recommended.
Input RNA (UHR) PS used in stride 17 Library* Insert Library yield PCR cycles
 Get-go [bp] Stop [bp] Mean size* Hateful size ≥ fifty 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
l 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 % vi % 2 % 1.viii  11.four 17
v ng 56 μl 100 1500 294 223 94 % 70 % 24 % seven % 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 v ng of loftier 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 (>v 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! Set up pre-warmed FS1 / FS2 / E1 mastermix! Place RNA samples at room temperature.
Footstep 3 Set FS2 / E1 mastermix – pre-warm for 2 – 3 minutes at 42 °C. Ready 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 together pre-warmed mastermix to RNA samples at room temperature.
OPTIONAL: Increase incubation time to 1 hour at 42 °C.
Step half dozen Incubate for 10 minutes at 95 °C. Incubate for x minutes at 95 °C.
Step 17 Add 56 μl of Purification Solution (PS). Reduce volume of Purification Solution (PS) to 48 μl.
Footstep 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 foreclose over- or undercycling of the libraries.
Step 29 Add together 36 μl of Purification Beads (PB). Reduce volume of Purification Beads (Pb) to 30 μl.

A further purification of the lane mix would be appropriate in order to remove all library fragments below 125 bp (inserts smaller than 41 bp). For more than information regarding the input RNA requirements please consult Appendix B (p. 23).

Yes, depression quality and FFPE samples can be used with QuantSeq. Some small protocol modifications are required though. These recommendations are indicated in the table below:
Protocol Step Standard Input (>v ng) Low quality / degraded / FFPE Input
Footstep 2 Incubate for iii minutes at 85 °C, then cool to 42 °C.
Concord samples at 42 °C on the thermocycler.		 Skip! Prepare pre-warmed FS1 / FS2 / E1 mastermix! Place RNA samples at room temperature.
Step 3 Set up FS2 / E1 mastermix – pre-warm for 2 – three minutes at 42 °C. Fix FS1 / FS2 / E1 mastermix – pre-warm for two – 3 minutes at 42 °C.
Step 4 Add pre-warmed mastermix to RNA / FS1 samples at 42 °C. Incubate for xv minutes at 42 °C. Add together 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 together 56 μl of Purification Solution (PS). Reduce book 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.one thousand., species, tissue, cell type)		 The qPCR assay is strongly recommended for all low input, FFPE / degraded RNA library preps, to preclude over- or undercycling of the libraries.
Step 29 Add 36 μl of Purification Beads (PB). Reduce book of Purification Chaplet (PB) to xxx μl.

The quality of RNA from FFPE tissues can vary greatly. We recommend measuring the DV 200 value (the per centum of RNA greater than 200 nt in length) in addition to RIN values, every bit RIN values go less meaningful for highly degraded samples.

Libraries prepared from FFPE RNA input typically crave different (often college) PCR bike numbers than those prepared with high quality RNA input (see tabular array below for examples). The DV 200 value is also not e'er 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 Bicycle Number
50 ng FFPE 15
10 ng FFPE eighteen
500 pg FFPE 22

* Please be aware the values in the table are guidelines but and are based on Mm brain FFPE RNA with a RIN of one.8 (DV 200 of 51 %). For different sources of RNA, and variable RNA qualities, more than (or less) PCR cycles might exist needed.

  • First Strand cDNA Synthesis
      1. At stride 3 pre-warm the FS2 / E1 mastermix for ii – 3 minutes at 42 °C while the RNA / FS1 samples are denaturing for 3 minutes at 85 °C – Do non cool the mastermix on ice!
      2. Afterward the RNA / FS1 samples have cooled to 42 °C, spin these down briefly and then immediately render 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 (pace 4) and mix properly. Whatever drib in temperature at this betoken can crusade mishybridization! Seal the plate or tubes and begin the 42 °C incubation.

    Notation! 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 v ul FS1, 9.5 ul FS2, and 0.5 ul E1, mix well, spin downward, 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 downwardly the pre-warmed FS1 / FS2 / E1 mastermix and add together 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)).
    • Keep immediately to the RNA removal after the reverse transcription is complete! Practise not place the samples on ice, and do non shop samples at -xx °C at this signal! Cooling the samples below room temperature at this point tin cause mishybridisation! All-time practise 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 x infinitesimal incubation at 95 °C (step half dozen).

NOTE! To minimise temperature drops at this indicate the reactions can also be kept at 42 °C while the RNA Removal Solution (RS) is added: Briefly spin downwardly the samples after pace 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 downwards, and place back on the thermocycler block and re-starting time the program for the 95 °C incubation.

  • When re-starting the protocol afterwards safe stopping points where libraries are stored at -twenty degrees, ensure that that they are thawed to room temperature before starting time the protocol once again. This is especially of import for purification steps as reduced temperatures here can impact bead migration and library precipitation, leading to a loss of yield.
Lexogen's QuantSeq kit is a library preparation protocol designed to generate fix-to-sequence Ion Torrent-compatible libraries from polyadenylated RNA within 4.5 hours. When you carry out the protocol for the first time please permit for more time and read the entire User Guide first.
QuantSeq libraries are intended for a loftier caste of multiplexing. Barcodes are introduced as in-line barcodes during second strand synthesis, allowing upward to 48 samples to be sequenced per lane. In-line barcodes are nine – 11 nt long followed by an additional 4 nt abiding 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 divergence is that nosotros consider the C nowadays in all barcodes a part of the constant region whereas IonTorrent counts the abiding C every bit the last nucleotide of the barcode and just the GAT as abiding part. When multiplexing QuantSeq libraries with IonXpress barcoded libraries brand certain to use unlike barcodes east.g., QuantSeq barcodes 1-48 with IonXpress barcodes 49-96.
The reads are straight reflecting the mRNA sequence and no re-orientation is necessary.

STAR aligner or bbmap tin be used for mapping. As QuantSeq is a 3' Seq protocol, virtually sequences will originate from the final exon and the three' untranslated region (UTR).

Alternatively TMAP mapping program can exist 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 outset 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 example information technology 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 get-go nucleotides tin 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 as well short or take more often than not low quality scores should be removed from the gear up.
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 however been fully evaluated).
In case of adapter contamination detection information technology is crucial to trim these sequences (east.m cutadapt, trim-gallore, or bbduk) in gild to align the reads.
The insert size is optimized for shorter reads (SR100). Withal, longer read lengths are besides possible if a more detailed assay of the very 3' terminate of transcripts is desired. Read lengths longer than SR100 can exist chosen (e.g., SR200, SR300, SR400), if the exact 3' cease is to exist pinpointed for nearly 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 top betwixt 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 drift at a lower speed on the Bioanalyzer chip or gels. This tin can interfere with verbal library quantification if relying solely on the Bioanalyzer results.
For futurity 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 effectually and beyond the upper marker of the Bioanalyzer. Make certain not to transfer any beads after the final elution in step 41 (Purification, QuantSeq User Guide, page 16). Get out approximately 2 µl of the eluate on the chaplet and do not endeavor to transfer the consummate sample, as this will lead to dewdrop carryover.
QuantSeq works fine with the HiQ organisation, however at that place 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 three' 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 (east.g., subsequently adding v µl in steps 5 and 7, employ a pipette set up to 15 µl or 20 µl for mixing).
  • Improver of the RS1 and RS2 solutions, they have to be added in an equal amount, otherwise you will become differences in the yield.
  • RS2 and SS1 have to be added in sequential club. 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 volition appear) equally this volition negatively influence the elution, but as well don´t carry over traces of EtOH to the side by side 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 expert positive command, the most of the reference values given in the User Guide are also based on UHRR input.

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