Description

QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

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

QuantSeq FWD contains the Illumina Read 1 linker sequence in the second strand synthesis primer, hence NGS reads are generated towards the poly(A) tail, directly reflecting the mRNA sequence (see workflow). This version is the recommended standard for gene expression analysis. Lexogen furthermore provides a high-throughput version with optional dual indexing (i5 and i7 indices) allowing up to 9,216 samples to be multiplexed in one lane.

Analysis of Low Input and Low Quality Samples

The required input amount of total RNA is as low as 100 pg. QuantSeq is suitable to reproducibly generate libraries from low quality RNA, including FFPE samples. See Fig.1 and 2 for a comparison of two different RNA qualities (FFPE and fresh frozen cryo-block) of the same sample.

correlation samples

Figure 1 | Correlation of gene counts of FFPE and cryo samples.

venn_diagrams

Figure 2 | Venn diagrams of genes detected by QuantSeq at a uniform read depth of 2.5 M reads in FFPE and cryo samples with 1, 5, and 10 reads/gene thresholds.

Mapping of Transcript End Sites

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

Figure 3 | QuantSeq read coverage versus normalized transcript length of NGS libraries derived from FFPE-RNA (blue) and cryo-preserved RNA (red).

Deplete globin mRNAs during QuantSeq Library Prep

New! Globin Block Modules for QuantSeq enable the generation of globin-depleted, ready-to-sequence 3’ mRNA-Seq libraries from as little as 50 ng of total RNA from whole blood. No additional protocol steps are required. The RS-Globin Block solution from the module simply replaces the standard RNA Removal Solution from the QuantSeq Kit. Using Globin Block for QuantSeq, reads mapping to globin mRNAs can be reduced from 50 – 80 %, to as low as 5% (Fig. 4) and gene detection is dramatically increased (Fig.5).
* For Figures: Input RNA (50 ng, or 250 ng) was extracted with: 1 SPLIT RNA Extraction Kit, 2 other kit including red blood cell lysis, or 3 other kit without red blood cell lysis.
Fig.1-Globin-Block_1000px

Figure 4 | Percentage of sequencing reads mapping to globin mRNAs from human (Hs) and pig (Ss) blood QuantSeq libraries.*

Fig.2-Globin-Block_1000px

Figure 5 | Increased gene detection in human blood QuantSeq libraries using Globin Block. CPM = Counts Per Million.*

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.

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.

Simple Bioinformatics Analysis

Read mapping is simplified by skipping the junction detection. Reads are generated at the transcripts’ most 3′ end where nearly no junctions are located. Data processing can hence be accelerated.

The QuantSeq data analysis pipeline has furthermore been integrated on the BlueBee genomics analysis platform and can be used by any user even without bioinformatics background. The access codes are included in QuantSeq kits. Additional codes can be purchased at the webstore. Promotion: until August 31, 2020 additional codes for up to 1.5 GB input are available free of charge.

Learn more and get started at https://www.bluebee.com/lexogen/. For using your activation code register an account with BlueBee (https://lexogen.bluebee.com/portal) and upload your data (fastq.gz files).

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. Up to 96 i7 indices are included in the single indexing kits (Cat. No. 015). Additional i5 indices can also be introduced using the Lexogen i5 6 nt Dual Indexing Add-on Kits (Cat. No. 047 or 015.384). Used together, Lexogen’s 96 i7 and 96 i5 6 nt indices enable up to 9,216 different index combinations for sequencing.

NEW! QuantSeq 3’ mRNA-Seq library prep is now also available with up to 384 pre-mixed Unique Dual Indices (Cat. No. 113, 114, 115). Lexogen’s new 12 nt UDIs feature superior error correction for maximal sequencing data output and are also available as stand-alone Add-on Kits (Cat. No. 107 – 111) for use with other library preps. For further questions, please contact support@lexogen.com.

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 (SR50, SR100).

Unique Molecular Identifiers

The UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1) (Cat. No. 081.96) contains the UMI Second Strand Synthesis Mix (USS). This mix simply replaces the Second Strand Synthesis Mix 1 (SS1) from the standard QuantSeq FWD Kit. The module allows unique tagging of individual transcripts with 6 nt long Unique Molecular Identifiers (UMI) located between the partial P5 adapter and the random priming sequence. Use this module to identify PCR duplicates and eliminate amplification bias.

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
Illumina-specific Read 2 linker sequence.
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 Illumina-specific Read 1
linker sequence. At this step Unique Molecular Identifiers (UMIs)
can be introduced by exchanging the Second Strand Synthesis Mix 1
(SS1) from the standard QuantSeq FWD Kit with UMI Second Strand
Synthesis Mix (USS).
04quantseq_workflowtime_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_workflowtime_quantseq05
Library Amplification
Step 4:
During the library amplification step sequences required
for cluster generation are introduced.
06quantseq_workflow_updtime_quantseq06
Library Amplification
Step 4:
Multiplexing can be performed with up to 9,216 barcode
combinations using the 96 available i7 indices and 96 i5 indices.
07quantseq_workflow_updtime_quantseq06
Sequencing
Step 5:
NGS reads are generated towards the poly(A) tail and directly
correspond to the mRNA sequence. To pinpoint the exact 3’ end,
longer reads may be required (SR50, SR100, SR150). Although
paired-end sequencing is possible, we do not recommend it
for QuantSeq FWD. Read 2 would start with the poly(T) stretch,
and as a result of sequencing through the homopolymer stretch,
the quality of Read 2 would be very low.

For viewing the whole workflow on page please click here

Featured Publications

Automation

autoQuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina

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 9,216 Illumina-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 protocol has been adapted for automated realization on the Sciclone NGS and Zephyr liquid handlers of PerkinElmer, the Hamilton Microlab STAR Workstations, the Agilent Bravo Automated Liquid Handling Platform and the Biomek platforms of Beckman Coulter. Contact us for implementation of QuantSeq on your automation platform.

Rapid Turnaround

Depending on the robot used the whole library preparation can be done in one day, including the manual preparation time. Since the individual protocol phases can be run on separate machines, further throughput enhancement can be achieved by parallelizing the workflow.

Easy Setup

An easy-to-follow Excel file guides you though preparation of all master-mixes and filling up the plates.

Flexibility of the Throughput

The QuantSeq kit is set up in a 96 well plate format and can generate libraries with up to 9,216 different barcode combinations. Depending on the liquid handler used you can either process any number of samples at the same time or need to run multiples of 8 reactions at once.

Avoiding Cross Contamination

The pre-PCR step and the post-PCR phase can be programmed on different machines which substantially reduces the risk of cross-contamination of the pre-PCR samples by the PCR products.

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.

Both kit versions yield sequences close to the 3’ end of transcripts. The difference is in the location of the Read 1 linker sequence. If it is located in the 5’ part of the second strand synthesis primer (QuantSeq Forward (FWD), Cat. No. 015), NGS reads will be generated towards the poly(A) tail (Figure 1). The FWD version is therefore recommended for all gene expression applications.

Figure 1 | Read orientation for QuantSeq FWD (Cat. No. 015).

With QuantSeq Reverse (REV, Cat. No. 016) the Read 1 linker sequence is located on the 5’ end of the oligodT primer and a Custom Sequencing Primer (CSP, included in the kit) is required for sequencing, in order to start the read directly at the 3’ end (Figure 2). Based on this the exact 3’ UTR can be pinpointed. REV is ideal for applications including alternative polyadenylation site identification and expression studies.

QuantSeq_faq02

Figure 2 | Read orientation for QuantSeq REV with CSP (Cat. No. 016).

The QuantSeq protocol was updated in February 2017 as follows:

  • Shortened RNA removal step as RS2 buffer is not used (RS1 was renamed to RS). Second strand synthesis Mix 1 (SS1), and second strand synthesis and purification step volumes were updated.
  • Barcodes in the i7 Index Plate were renamed (7001-7096, formerly BC01-96) and rearranged column-wise for improved nucleotide balance for sequencing when only few samples are multiplexed. BC05 was replaced by i7 index 7025 to ensure the full set are unique from Illumina TruSeq indices.
  • The QuantSeq FWD HT Kit (015.384) was released at the same time, which furthermore provides a high-throughput version with optional dual indexing (i5 and i7 indices) allowing up to 384 barcode combinations (QuantSeq 3’ mRNA-Seq FWD HT User Guide).
The QuantSeq protocol is optimized for shorter reads (SR50 – 100) and yields mean library sizes of about 335 – 456 bp, with mean insert sizes of 203 – 324 bp. To generate longer libraries, use the QuantSeq-Flex First Strand Synthesis Module (Cat. No. 026), see QuantSeq 3’mRNA-Seq User Guide, Appendix G, p.28.
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, animal and plant tissues, yeast, fungi, drosophila and human reference RNA.
As a starting point, we recommend performing the protocol initially with 500 ng total RNA. RNA inputs ≥200 ng are recommended to detect low abundant transcripts efficiently.
The minimum recommended input amount is 100 pg for high quality total RNA. Protocol modifications apply for low input (<= 10 ng) and low quality or FFPE RNA (see FAQ 1.5 below, and Appendix C of the QuantSeq User Guide, p.23).
QuantSeq FWD was successfully tested with as little as 10 pg of Universal Human Reference (UHR) RNA input. When using <10 ng, or <1 ng of total RNA input please follow the recommendations indicated in the table below:

Protocol Step Standard Input (>10 ng) Low Input (≤10 ng) * FFPE / Degraded RNA Low Input (≤1 ng) *
Step 1 Add FS1 to RNA samples. Do not place samples back on ice after adding FS1! Skip! Place RNA samples briefly at room temperature while the mastermix is prepared.
Step 2 Incubate for 3 minutes at 85 °C, then cool to 42 °C.
Hold samples at 42 °C on the thermocycler.
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 on the thermocycler at 42 °C. Incubate for 15 minutes at 42 °C. Add pre-warmed mastermix to RNA samples at room temperature and transfer to a thermocycler preheated to 42 °C. Incubate for 15 minutes at 42 °C, or increase incubation time to 1 hour.
Step 6 Incubate for 10 minutes at 95 °C. Incubate for 10 minutes at 95 °C. Incubate for 5 minutes at 95 °C.
Step 16 Add 56 μl of Purification Solution (PS). Reduce volume of Purification Solution (PS) to 48 μl.
Step 24 The qPCR assay is strongly recommended for optimizing the number of PCR cycles required for library amplification. This will prevent under- or overcycling of the libraries (see Appendix E, p.25). The qPCR assay should be performed also when RNA samples are of:
• Variable input amounts
• Variable quality (RIN / RQN) or purity (absorbance ratios: 260/280 and 260/230)
• Variable type (e.g., species, tissue, cell type)
• FFPE origin, or highly degraded
Step 29 Add 30 μl of Purification Beads (PB) for single-indexed libraries, or 35 μl for dual-indexed libraries. Reduce volume of Purification Beads (PB) to 27 μl for single-indexed libraries, or 31.5 μl for dual-indexed libraries.

* Only for QuantSeq FWD. Minimum input for QuantSeq REV is 10 ng.

The number of PCR cycles optimal for a given input amount of total RNA can vary by up to four cycles, depending on sample quality and origin. The optimal cycle number for your specific sample type should be determined using the qPCR assay (see below, and Appendix E, p.25).

The table below is provided as a reference only! Optimal cycle numbers could exceed these ranges depending on the sample type (i.e., species, tissue, RNA quality e.g., FFPE RNA, etc).

Total RNA Input Amount No. Cycles for Endpoint PCR **
0.5 ng * 21 – 25
10 ng * 17 – 20
100 ng 14 – 17
≥500 ng 11 – 14

*Indicates preparation using low input protocol modifications with 1 hour incubation at 42 °C at step 4.
**These values are provided as a reference only! Sample type influences the optimal cycle number which should be determined by qPCR assay.

The qPCR assay to determine optimal PCR cycle numbers for library amplification is performed using an aliquot of purified double-stranded cDNA produced at step 24. The PCR Add-on Kit (Cat. No. 020.96), and SYBR Green I nucleic acid stain (10,000X in DMSO, not provided in the kit. We recommend using Sigma-Aldrich Cat. No. S9430) are required for this qPCR assay.

ATTENTION: The use of SYBR Green I-containing qPCR mastermixes from other vendors is not recommended.

The recommended protocol for the qPCR assay is outlined in Appendix E, p.25 of the QuantSeq 3’ mRNA-Seq Library Prep Kit User Guide.
Briefly, the cDNA obtained at step 24 is diluted with EB to a total volume of 19 μl and a 1.7 μl of this cDNA is then used as template for a qPCR containing PCR Mix (PCR), Enzyme Mix 3 (E3), P7 primer (7000), and SYBR Green I dye at a final concentration of 0.1x (NOTE: Higher concentrations of SYBR Green I can inhibit amplification). PCR is performed in a real-time PCR machine for 35 cycles and the amplification curves (in linear scale) are then used to determine the cycle number corresponding to 50 % of the maximum fluorescence reached at the amplification plateau (See Fig. 3, below). Given that the remaining volume of cDNA for endpoint PCR is ~17 μl, which is 10x more than the 1.7 μl used for the qPCR assay, 3 cycles are subtracted from this number to give the optimal endpoint PCR cycle number.

Calculation_of_the_number_of_cycle_for_the_endpoint_PCR

Figure 3 | Calculation of the number of cycles for the endpoint PCR. For this sample, 50 % of the maimum fluorescence at the amplification plateau corresponds to 15 cycles. As endpoint will use 17 μl of cDNA as template (10x more than used for qPCR) 3 cycles are subtracted givin 12 cycles to use for endpoint PCR.

The PCR Add-on Kit (Cat. No. 020.96) can be used to perform a qPCR assay for calculating the optimal number of PCR cycles to use for Endpoint PCR. This assay can be used also when dual indexing is intended.

The PCR Add-on Kit cannot be used for reamplification of dual-indexed libraries. Instead, the Reamplification Add-on Kit for Illumina (Cat. No. 080.96) is required and can be obtained from Lexogen upon request via support@lexogen.com.

Yes, low quality and FFPE samples can be used with QuantSeq. Some minor protocol modifications are required and are indicated in the table below:

Protocol Step Standard Input (>10 ng) Low Input (≤10 ng) * FFPE / Degraded RNA Low Input (≤1 ng) *
Step 1 Add FS1 to RNA samples. Do not place samples back on ice after adding FS1! Skip! Place RNA samples briefly at room temperature while the mastermix is prepared.
Step 2 Incubate for 3 minutes at 85 °C, then cool to 42 °C.
Hold samples at 42 °C on the thermocycler.
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 on the thermocycler at 42 °C. Incubate for 15 minutes at 42 °C. Add pre-warmed mastermix to RNA samples at room temperature and transfer to a thermocycler preheated to 42 °C. Incubate for 15 minutes at 42 °C, or increase incubation time to 1 hour.
Step 6 Incubate for 10 minutes at 95 °C. Incubate for 10 minutes at 95 °C. Incubate for 5 minutes at 95 °C.
Step 16 Add 56 μl of Purification Solution (PS). Reduce volume of Purification Solution (PS) to 48 μl.
Step 24 The qPCR assay is strongly recommended for optimizing the number of PCR cycles required for library amplification. This will prevent under- or overcycling of the libraries (see Appendix E, p.25). The qPCR assay should be performed also when RNA samples are of:
• Variable input amounts
• Variable quality (RIN / RQN) or purity (absorbance ratios: 260/280 and 260/230)
• Variable type (e.g., species, tissue, cell type)
• FFPE origin, or highly degraded
Step 29 Add 30 μl of Purification Beads (PB) for single-indexed libraries, or 35 μl for dual-indexed libraries. Reduce volume of Purification Beads (PB) to 27 μl for single-indexed libraries, or 31.5 μl for dual-indexed libraries.

* Only for QuantSeq FWD. Minimum input for QuantSeq REV is 10 ng.

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 are typically more variable, hence optimization of PCR cycle numbers is highly recommended. To prevent under- or overcycling the libraries, we 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. 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.

NOTE: The DV200 value is not always a reliable predictor of the required number of PCR cycles needed.

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.

Lexogen’s QuantSeq kit is a library preparation protocol designed to generate sequence-ready Illumina-compatible libraries from polyadenylated RNA within 4.5 hours. When carrying out the protocol for the first time, please allow for more time, including time for performing the qPCR assay.

Read the entire User Guide before starting and ensure all kit components are prepared according to specified instructions.

QuantSeq libraries are intended for a high degree of multiplexing. Indices are introduced as standard external barcodes during the PCR amplification step. With the up to 96 i7 indices (Lexogen i7 6 nt Index Plate, 7000-7096) included in the kit and additionally available i5 indices (5001-5009, Lexogen i5 6 nt Dual Indexing Add-on Kits, Cat. No. 047), up to 9,216 samples can be prepared with different i5 / i7 index combinations, for multiplexed sequencing in a single lane (or run) on Illumina instruments.

All i7 and i5 indices are 6 nucleotides (6 nt) in length. Sequences are available from the Downloads section.

Please note that four i5 indices are already included in QuantSeq FWD HT (5001 – 5004, Cat. No. 015.384).

Multiplexing QuantSeq libraries with other library types in the same sequencing lane is not recommended. For further information please see FAQ 1.30.

QuantSeq FWD (Cat. No. 015) generates NGS reads towards the poly(A) tail. To pinpoint the exact 3’ end, longer read lengths may be required. Read 1 directly reflects the mRNA sequence.
STAR aligner is recommended for mapping QuantSeq FWD (Cat.No. 015) data. The reads may not land in the last exon and span a junction. Reads should be trimmed prior to alignment to remove poly(A) tails and sequencing adapters (see FAQ. 1.13 below).
More information on the data analysis can be found here.
The reads should be trimmed to remove adapter sequences, poly(A) / poly(T) sequences, and low quality nucleotides. Reads that are too short (i.e., <20 nt) or have generally low quality scores should be removed from the set.

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. For QuantSeq FWD data we therefore recommend using an aligner that can perform soft-clipping of the read ends (e.g., STAR aligner) during alignment, or increasing the number of allowed mismatches to 14. Alternatively, trimming the first 12 nt of Read 1 can be performed prior to alignment when using a more stringent aligner (e.g., HISAT2). While trimming the read can decrease the number of reads of suitable length for alignment, the absolute number of mapping reads may increase due to the improved read quality.

The QuantSeq 3’ mRNA-Seq FWD kit is appropriate for GAIIX, HiSeq 2000/2500/300/4000, MiSeq, NextSeq 500/550, MiniSeq, and NovaSeq Illumina platforms.
For most applications single-read 50 or 75 (SR50, SR75) is sufficient for QuantSeq FWD sequencing.

When UMIs are included in QuantSeq FWD libraries, the minimum recommended sequencing length is SR75 (see online FAQ. UMI Specific 4.6).

To increase the number of uniquely mapping reads or to better resolve exact 3’ ends by reading into the poly(A) tail, longer reads may be used (SR100, SR150).

Transcripts may have different and not yet annotated 3’ ends, which might be mistaken for internal priming events of the oligodT primer, when in fact those are true 3’ ends. Artificial spike-in transcripts such as the SIRVs or the ERCC spike-in transcripts only have one defined 3’ end, this provides the only true measure to determine internal priming.

If true internal priming is detected, this could be a result of mis-priming during reverse transcription, for instance if the temperature before or during reverse transcription was too low. As outlined in the general section of the User Guide, unless explicitly mentioned, all steps should be carried out at room temperature between 20 °C and 25 °C. In particular, for First Strand cDNA Synthesis (p.11-12): Do not cool the FS2/E1 mastermix (step 3) and have the RNA/FS1 samples at 42 °C when adding the FS2/E1 mastermix (step 4) to avoid mishybridization. Mix properly by pipetting. Do not forget to shortly spin down the samples at room temperature before and after adding the FS2/E1 mastermix.

ATTENTION: Centrifugation should not be carried out at 4 °C, always spin down at room temperature! Raising the reaction temperature up to 50 °C for reverse transcription can be used for additional prevention of mis-priming.

  • First Strand cDNA Synthesis (p.11-12):
    1. Prepare your RNA if 5 μl volumes and bring to room temperature for 2-5 minutes before adding the FS1 buffer. Ensure FS1 is also fully equilibrated to room temperature. ATTENTION! Do not place RNA / FS1 samples back on ice and proceed immediately to denaturing after FS1 is added.
    2. 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!
    3. 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.
    4. 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.
    5. After the 42 °C incubation is complete, proceed immediately to RNA Removal. Do not cool the samples on ice, or store below room temperature at this point!

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

  • If steps 1 and 2 (denaturing) are skipped (low input or degraded samples i.e. ≤10 ng, or FFPE samples):
    1. Prepare your RNA samples in 5 μl volumes.
    2. Prepare a mastermix containing 5 μl FS1, 9.5 μl FS2, and 0.5 μl 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 for 2-5 minutes while the mastermix is pre-warming. Adding mastermix to cooled RNA samples can result in mishybridization!
    4. Spin down the pre-warmed FS1 / FS2 / E1 mastermix and add 15 μl 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 on a thermocycler (or 1 hour for low input RNA (≤ 10 ng)).
    5. After the 42 °C incubation is complete, proceed immediately to RNA Removal. Do not cool the samples on ice, or store below room temperature at this point!
  • Proceed immediately to the RNA removal after the reverse transcription is complete! (step 4 – 5). The previous safe stopping point after reverse transcription (step 4) has been removed. 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.

A second peak between 1,000–9,000 bp, or an elevated baseline on a bioanalyzer trace (or similar microcapillary electrophoresis trace) is an indication of overcycling (Figure. 4). (For another example of how overcycled libraries can look in general, see also SENSE mRNA FAQ 1.10).

Correct-versus-Overcycled-QuantSeq-Library-Results

Figure 4 | Correct versus Overcycled QuantSeq Library Results: Correctly cycled (10 ng UHRR 19 cycles, blue), Overcycled (10 ng UHRR, 24 cycles, red). Input protocol modifications (skipping step 2, 1 hour incubation at 42 °C at step 4, reduced volumes of PB and PS at steps 16 and 29).

Overcycling occurs when too many PCR cycles are used for the final library amplification, when the PCR runs out of primers and template and generated ds-cDNA starts 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 for pooling and loading for sequencing.

A qPCR assay for exact library quantification should be used in addition to Bioanalyzer or similar results, if high molecular weight peaks occur and overcycling is supsected.

Overcycled libraries may have more PCR duplicates which can affect quantification accuracy and sample clustering (i.e by PCA).

For future QuantSeq library preps:

  • Perform the qPCR assay to determine the optimal number of PCR cycles required for library generation.
  • For similar samples reduce the number of PCR cycles for library amplification (Endpoint PCR) to prevent overcycling.

Elevated baseline above the upper marker on Tapestation traces can indicate overcycling (see FAQ 1.18 above for further details).

A carryover of Purification Beads (PB) can also produce a peak around and beyond the upper marker of the Tapestation and also on Bioanalyzer. Make sure not to transfer any beads after the final elution in step 41 (or 42 for dual indexing). A few tips:

  • Leave approximately 2 µl of the eluate on the beads when removing the eluate for QC and sequencing. i.e., Transfer only 15-17 μl of the eluate into a fresh PCR plate or tube.
  • Do not try to transfer the complete sample, as this will lead to bead carryover.
  • Put your samples once again on the magnet when taking an aliquot to run for QC on Tapestation/Bioanalyzer/Fragment Analyzer, or similar.

In many cases, undercycled libraries will still contain sufficient amounts of cDNA for pooling and sequencing and will not need to be reamplified.
To check whether your library amounts are sufficient for sequencing, input the measured concentrations to the Library Quantification Calculation File and adjust the amount of each library to be pooled (minimum = 10 fmol). So long as the volume indicated for pooling is below the volume of cDNA available the library can be sequenced.

Should amounts of undercycled libraries be insufficient for sequencing, the PCR Add-on Kit for Illumina (Cat. No. 020) includes a Reamplification Primer that can be used to add some PCR cycles for your undercycled libraries (QuantSeq 3’ mRNA-Seq User Guide, Appendix E: qPCR and Reamplification, p.25). Please note that currently only single indexed libraries can be reamplified using the PCR Add-on Kit (Cat. No. 020). Dual-indexed libraries can be reamplified only using the Reamplification Add-on Kit for Illumina (Cat. No. 080.96) which is available upon request by contacting support@lexogen.com.

We recommend using Universal Human Reference RNA (UHRR, Agilent Technologies) as a positive control.
Paired-end (PE) sequencing is typically not recommended for QuantSeq FWD (Cat. No. 015), as the quality of Read 2 is very low due to the poly(T) stretch at the beginning of Read 2. However, if QuantSeq FWD lane mixes are sequenced in PE mode, we recommend discarding Read 2 data and proceeding with Read 1 data only for downstream data analysis (i.e., use only Read 1 for trimming, alignment, read counting, and downstream analyses).

For PE sequencing, QuantSeq REV (Cat. No. 016) may also be used. Please note QuantSeq REV requires a Custom Sequencing Primer (CSP) for Read 1, in order to generate reads that begin at the exact 3’ end of the transcript.

To generate longer libraries, use the QuantSeq-Flex First Strand Synthesis Module (Cat. No. 026).

Briefly, mix 5 μl of RNA with 5 μl of the oligodT primer provided in the QuantSeq-Flex First Strand Synthesis Module, and denature for 3 minutes at 85 °C, then cool the samples to hold at 42 °C (leave them on the thermocycler). Prepare a mastermix of FS1x 5 μl, FS2x 4.5 μl, and E1 0.5 μl per sample, and pre-warm at 42 °C for 2-3 minutes. Then add the pre-warmed mastermix to the RNA / oligodT samples while keeping the samples on the thermocycler incubating at 42 °C. Mix and spin down quickly then return the samples to the thermocycler to incubate for 15 minutes at 42 °C. Changing the denaturing conditions extends the library insert size (Figure 5).

Fig_6_QuantSeq_FWD

Figure 5 | Bioanalyzer traces of QuantSeq FWD (std.) and Flex libraries prepared from 500 ng UHRR input RNA. Input RNA was denatured for 3 minutes at 85 °C, with either 5 μl oligodT from the QuantSeq-Flex First Strand Synthesis Module (Cat. No. 026; blue trace, RNA+dT), or the standard QuantSeq FWD FS1 buffer (red trace, std.). Average library size is increased when RNA+dT conditions are used. Libraries were amplified with i7 and i5 6 nt index primers, using Dual PCR Mix (Lexogen i5 6 nt Dual Indexing Add-on Kit, Cat. No. 047) and 13 PCR cycles.

More information can be found in the QuantSeq 3’mRNA-Seq FWD User Guide, Appendix H: Modulating Insert Sizes, p.30.

The loading amounts indicated below are provided a guideline for sequencing QuantSeq FWD/REV libraries in pure pools for each instrument. Multiplexing QuantSeq with other external library types is not recommended (see FAQ 1.30). We therefore cannot provide optimal pooling or loading amount guidelines for lanemixes that include QuantSeq libraries in diverse multiplexing setups.

Loading amounts (and PhiX spike in percentages) may still need to be adjusted to obtain optimal cluster densities for your respective instrument, or if sequencing chemistries are changed. Inaccuracies in library quantification can affect the loading amount required. Please ensure accurate quantification of lane mixes and lane mix dilutions when preparing for loading.

Unless otherwise indicated the values given in the table below are based on customer experiences. For further inquiries regarding loading amounts please contact support@lexogen.com.

Sequencer QuantSeq FWD (Cat. No. 015),
PhiX: 1 – 5 %
QuantSeq FWD with UMIs (Cat. No.s 015/081),
PhiX: 5 – 30 %
QuantSeq REV (Cat. No. 016),
PhiX: none *
HiSeq 3000 / HiSeq 4000 / HiSeqXTen 280 – 350 pM 1 250 – 350 pM (PhiX: 5 %) **
HiSeq 2000 / HiSeq 2500 10 pM ** 10 – 12.5 pM
MiSeq 6 – 15 pM ** 6 – 15 pM
MiniSeq 1.3 – 1.8 pM ** **
NextSeq 500 / 550 2 – 2.5 pM 2 1.8 pM 3 (PhiX: 15 – 30 %) **
NovaSeq 6000 Standard: 300 – 500 pM 4 , Xp: 325 – 400 pM 5 ** **

* PhiX cannot be sequenced in QuantSeq REV runs due to the use of a custom sequencing primer instead of the Multiplex Read 1 Sequencing Primer, which does not bind to PhiX.
** Please inquire at support@lexogen.com..
1 Loading amounts are based on customer-supplied feedback for optimal sequencing quality on HiSeq 4000.
2 The values for NextSeq 500 / 550 are recommended loading amounts and have been evaluated by Lexogen to ensure optimal cluster densities of 200 – 260 K/mm2 are achieved. Loading amounts below 2 pM for QuantSeq FWD libraries may result in lower cluster densities and reduced index read quality.
3 Loading amounts for QuantSeq FWD-UMI libraries are reduced compared to FWD libraries without UMIs, and PhiX must be spiked in at a minimum of 15 % for NextSeq runs, due to the presence of a low diversity spacer sequence at positions 7-10 of each library insert. This is to maintain higher per base quality for these cycles, and PF rates.
4 Loading amounts for NovaSeq Standard workflow are based on user experience using S1 and S2 flow cells, and should be relevant for all flow cell types. This range is further calculated according to Illumina’s recommendation to use 1.5x the loading concentrations for HiSeq 4000. The calculated loading amounts shown fall within the Illumina recommended range for PCR-amplified library pools (300 – 600 pM).
5 Loading amounts for NovaSeq Xp workflow are based on customer-supplied feedback from sequencing of SENSE mRNA V2 libraries. These concentrations also relate to customer-validated loading amounts used for HiSeq 4000 instruments.

In general, we recommend processing a minimum of 8 samples, using a complete set of eight i7 indices for multiplexing (e.g., 7001-7008). However, if fewer barcodes are required care should be taken to always use indices which give a well balanced signal in both lasers (red and green channels) for each nucleotide position. All columns (1-12) and rows (A-H) fulfill these criteria. The individual libraries within a lane should be mixed at an equimolar ratio to ensure this balance. An evaluation tool to check the color balance of index subsets is available under Support Tools.
The QuantSeq FWD HT kit is based on the standard QuantSeq FWD kit but contains enough reagents for 384 library preparations with unique barcode combinations through the use of the integrated Lexogen i5 6 nt Dual Indexing Add-on Kit.
Yes! All QuantSeq FWD and REV kits ordered after November 2016 are supplied with a code to access the QuantSeq data analysis pipelines on the BlueBee® Genomics Platform.

The code supplied with the QuantSeq FWD Kits (Cat. No. 015) enables two different pipeline options:

FWD pipeline – For standard QuantSeq FWD library data (no UMIs).
FWD-UMI pipeline – only for libraries prepared with the UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1, Cat. No. 081).

Each code contains an equal number of pipeline runs as reactions provided in the kits, i.e. you should choose either the FWD or FWD-UMI pipeline, depending on how your libraries were prepared. If you wish to run both options, additional Activation Codes will need to be purchased (Cat. No. 090). If you start the wrong pipeline for your data you can abort or stop the run before it is completed without losing your allocated runs. If the run is completed then additional runs will need to be purchased.

To analyze data from QuantSeq FWD libraries that contain UMIs (prepared with the UMI Second Strand Synthesis Module (Cat. No. 081), simply use the activation code included with your QuantSeq FWD kit and select the respective “FWD-UMI” pipeline when setting up your data analysis run on BlueBee® (for further information see FAQ 4.7 and 4.10).

QuantSeq FWD libraries prepared from blood using Globin Block (RS-GBHs or RS-GBSs, Cat. No. 070 and 071) should be analysed using the standard FWD pipeline for data analysis; unless UMIs are also included and then the FWD-UMI pipeline should be selected.

NOTE! Do not run the “FWD-UMI” pipelines for QuantSeq FWD (standard) libraries do not contain UMIs. This will incorrectly collapse reads and result in incorrect read count data. Use only the FWD pipelines for standard QuantSeq FWD libraries!
Be sure to only select the “FWD-UMI” pipelines for UMI-containing libraries, otherwise duplicate reads will not be collapsed.

ATTENTION: Data Analysis access is only free using the provided code, for fastq input files up to 1.5 GB in size. Larger fastq file sizes can only be processed using activation codes for large file sizes, which can be purchased additionally (Cat. No. 093 for FWD, or Cat. No. 094 for REV library types, respectively).

Do not use activation codes provided with the QuantSeq REV Kit (Cat. No. 016) for analysis of FWD libraries.

Analysis pipelines for QuantSeq (FWD, FWD-UMI, and REV) are currently available for the following species:

Common name (if available) (Species)

  • African Oil Palm (Elaeis guineensis)
  • Arabidopsis Thale cress (Arabidopsis thaliana)
  • Baker’s Yeast (Saccharomyces cerevisiae)
  • Brain-Eating Amoeba (Naegleria fowleri)
  • Chicken (Gallus gallus)
  • Cow (Bos taurus)
  • Common yellow monkeyflower (Mimulus guttatus)
  • Daphnia (Daphnia pulex)
  • Dog (Canis lupus familiaris)
  • Drummond’s Rockcress (Boechera stricta)
  • Ferret (Mustela putorius furo)
  • Fruit Fly (Drosophila melanogaster)
  • Fungus (Fusarium oxysporum)
  • Goat (Capra hircus)
  • Human (Homo sapiens)
  • Maize/Corn (Zea mays)
  • Melon Fly (Bactrocera cucurbitae)
  • Mouse (Mus musculus)
  • Nematode Roundworm (Caenorhabditis elegans)
  • Painted Turtle (Chrysemys picta bellii)
  • Pig (Sus scrofa)
  • Poplar (Populus poplar)
  • Potato (Solanum tuberosum)
  • Purple False Brome (Brachypodium distachyon)
  • Rabbit (Oryctolagus cuniculus)
  • Rat (Rattus norvegicus)
  • Rice (Oryza sativa)
  • Salmon (Salmon salar)
  • Sorghum (Sorghum bicolor)
  • Starlet Sea Anemone (Nematostella vectensis)
  • Tomato (Solanum lycopersicum)
  • Yeast (Candida albicans)
  • Yeast (Kluyveromyces lactis)
  • Zebrafish (Danio rerio)

New species pipelines can be added upon request for an additional fee. Please contact support@lexogen.com if your species of interest is not listed, and for pricing information.

All QuantSeq FWD and REV kits ordered after November 2016 are supplied with an activation code to access the QuantSeq Data Analysis pipelines on the BlueBee® Genomics Platform.

The activation codes are printed on a sticker that is attached to the side of the small reagent box, (stored at -20 °C) which comes inside the main QuantSeq Kit box (see Figure 6). Activation codes for additional runs can be purchased from Lexogen via the webshop. Activation codes registered after September 20, 2018 are valid for two years. The input file size is limited to 1.5 GB per fastq(.gz) file. If you have larger input files or for further inquiries, please contact support@lexogen.com.

Bluebee_Sticker_Location
Figure 6 | Location of the BlueBee® Data Analysis activation code on QuantSeq Kit reagent boxes.

The count summary files from the results of the data analysis pipeline on BlueBee® contain mapped read counts for Ensembl gene IDs. Ensembl gene IDs can be converted into other gene ID types, or additional annotations added using the BioDBnet tools: https://biodbnet-abcc.ncifcrf.gov/db/db2db.php. Simply input the list of Ensembl gene IDs, select the input format as “Ensembl Gene ID”, and then select the output format(s) desired.
We do not recommend multiplexing Lexogen libraries with other library types in the same sequencing lane. Though this is possible in principle, specific optimization of index combinations, library pooling conditions, and loading amounts may be required, even for advanced users. Sequencing complex pools that include different library types at different lane shares may have unpredictable effects on sequencing run metrics, read quality, read outputs, and/or demultiplexing performance. Lexogen assumes no responsibility for the altered performance of Lexogen libraries sequenced in combination with external library types in the same lane (or run).

Due to size differences, libraries prepared with the Lexogen Small RNA-Seq Library Prep Kit (or any other small RNA library prep kit) should not be sequenced together with QuantSeq, SENSE, or CORALL libraries. Please refer to the sequencing guidelines for each library type (library adapter details, loading amounts to use, and use of custom sequencing primers, etc), which are provided in our Library Prep Kit User Guides, and online Frequently Asked Questions (FAQs).

The autoQuantSeq 3′ mRNA-Seq protocol is currently automated on the following liquid handling instruments:

  • PerkinElmer Sciclone / Zephyr
  • Hamilton Microlab STAR / STARlet / NGS STAR (in progress)
  • Agilent Bravo NGS Workstation (B)
  • Beckman Coulter Biomek FxP and i7
  • Eppendorf EpMotion 5075

More information about QuantSeq Automation on Agilent Bravo can be found in our QuantSeq Automation Application Note.

Further information for Automation on Beckman FxP can also be obtained from the Application Overview Flyer.

Please contact support@lexogen.com if you are interested in automating QuantSeq on additional liquid handling platforms, or to obtain further information.

Script files for QuantSeq Automated protocols are available for all major liquid handers as listed in 2.1. Please note that modification of the program often required to ensure that the protocol runs optimally on your specific instrument and depends on the deck setup. Please contact support@lexogen.com and let us know which specific instrument you are using and connect us with your local Field Application Specialist, and we will advise you how to obtain the most relevant program script (see also FAQ 2.4, below).

Note: Automation scripts are provided for the full library prep protocol. Automating only the post-PCR purification can also be achieved by modifying an existing a PostPCR SPRI bead purification program. Please ensure the double bind (steps 32 and 34 are included).

Depending on your deck setup the automated protocol can be adapted for preparing up to 96 samples in parallel.

An existing protocol for the Hamilton STAR is designed to process up to 48 samples at once. Existing protocols for the Agilent Bravo NGS Workstation (B), and Beckman Coulter Biomek FxP and i7, can process up to 96 samples in parallel. The program for Agilent´s Bravo runs multiples of 8 reactions (i.e. column-wise from columns 1-12).

For the PerkinElmer Sciclone, also runs reactions in multiples of 8 samples (column-wise). Any number of columns from 1 to 12 can be run in parallel. The machine will always take the barcodes from the first N wells of the barcode plate, starting with column 1.

The program for Perkin Elmer Zephyr is only available for the purification steps. Please use the protocol for the Perkin Elmer Scicloe for the Phase1-PrePCR automated protocol.

The protocol script files that can be provided have been developed to run on the softwares and instrument types specified by each manufacturer. However, software version compatibility, and deck compatibility should always be checked first by the manufacturer of your instrument to ensure that the script will run correctly. It is usual that some optimization is required. We can supply dummy reagents for optimising liquid handling steps (Cat. No. 019.384, shipping charges apply). Please contact us for further information via support@lexogen.com.

We recommend the following process when adopting automation of any of our library prep protocols:

  • Contact the Field Application Specialist (FAS) or manufacturer of your liquid handler and ask them to check the compatibility of your instrument with the latest version of the automation script protocol.
  • After installing the program, perform a dry run to ensure there are no errors or incompatibilities.
  • Perform a dummy run using dummy reagents and check volume accuracy. We strongly recommend performing a dummy run using real purification reagents. Additional Magnetic Bead Purification Modules can be purchased for this purpose (Cat. No. 022, please enquire with your distributor or sales representative for pricing).
  • Perform a test run for a small number of replicates, using reference RNA (e.g., Universal Human Reference RNA, Agilent Technologies, Cat. No. 740000).
  • Contact our Tech Support Team (support@lexogen.com) for feedback and to enquire about the need for further optimization.
This option is up to the user. Using an on-deck thermocycler provides a higher level of automation compared to an off-deck thermocycler. However, both options will require manual interventions. The use of on-deck thermocyclers may not be possible with all instrument types, and this must also be specified in the automation script protocol.

If a stand-alone thermocycler is available in a comfortable vicinity of the robot, we can suggest to use an off-deck thermocycler for steps 2-3, and step 4, as conventional thermocyclers provide heated lid options that prevent condensation. Depending on the type of on-deck thermocycler used, a heated lid option may not be provided.

The current Perkin Elmer Sciclone, Eppendorf EpMotion 5075, Hamilton STAR / STARlet / NGS STAR, and Agilent Bravo Workstation (B) protocols are setup for off-deck thermocyclers. The current Beckman Coulter Biomek FxP/i7 protocols are written for on-deck thermocycling at all steps, including the PCR. However, modifications to all protocols can be made in collaboration with the respective liquid handler manufacturer.

Yes, if thermocycler incubations are run off-deck, five such interventions (including PCR) are required. The machine stops and asks the operator to do various steps such as, e.g., take the plate with samples, seal it with film, do the incubation step(s) on the external thermocycler, unseal the plate, place it on deck again, and resume the run.

To simplify the manual preparation, the Elution Buffer (EB) is presented in a 50 ml reservoir (trough). Robotic aspiration from this type of vessel requires a higher dead volume. Therefore, the amount of the EB required for automation might exceed the amount delivered in the kit. If this is the case, the EB can simply be substituted by 10 mM TRIS pH 8.0.

The QuantSeq 3’ mRNA-Seq kits contain enough components to generate up to 96 reactions for manual or automated library preparation. Therefore, there should be sufficient reagent to run all reactions at once on your liquid handler. However, if you want to split your kit into several machine runs you might loose a few reactions due to the higher dead volume needed compared to the manual preparation. Please contact us via support@lexogen.com if you experience reagent shortages.

Many liquid handlers do not have additional heat block positions on deck to warm mastermixes. However, the most critical step for QuantSeq first strand cDNA synthesis is that the RNA / FS1 samples are kept continuously at 42 °C after denaturing (step 2), and while the FS2 / E1 mastermix is added. Although pre-warming this mastermix helps to prevent mishybridisation by maintaining the sample temperature of the reactions after denaturing, the pre-warming can be omitted for automated protocols as long as the FS2 / E1 mastermix is prepared in advance and kept at room temperature on deck. This mastermix should not cooled or kept on a refrigerated block on deck prior to adding to the samples (at step 3).

No. QuantSeq FWD and REV libraries are prepared using the same protocol steps and reaction volumes. The use of Globin Block (RS-GBHs/RS-GBSs) or UMI Module (USS), is performed by exchanging these module solutions with the standard RNA Removal Solution (RS), or Second Strand Synthesis Mix 1 (SS1) at the respective steps (5 and 7). The volume of addition is the same at each step so only the supplied reagent tube needs to be exchanged – no protocol or volume changes are required. Therefore, the same automation protocol can be used for preparing all types of QuantSeq FWD and REV libraries, with or without Globin Block and / or UMIs.

Please note QuantSeq Flex libraries may require different automated protocol modifications depending on the use of custom primers at either or both of first and second strand synthesis steps of library generation. QuantSeq Flex programming is available for the NGS Workstation B (Agilent Bravo). However, please contact support@lexogen.com if you plan on automating QuantSeq Flex on any liquid handling platforms.

The Globin Block Modules can only be used with the QuantSeq 3’ mRNA-Seq Kits. They are compatible with both the FWD (Cat. No. 015) and REV (Cat. No. 016) versions of the kit and can also be used with the kits for Ion Torrent (Cat. No. 012). For information on the use of Globin Block Modules with the QuantSeq 3’ mRNA-Seq Library Prep Kit for Ion Torrent protocol, please contact support@lexogen.com.

The Modules are not compatible with the SENSE mRNA-Seq V2 (Cat. No. 001), SENSE Total RNA-Seq (Cat. No. 009), or CORALL Total RNA-Seq Library Prep Kits (Cat. No. 095, 096).

NOTE: These Globin Block Modules are not intended for use with any other external library prep protocols.

Yes. When using the modules with the older version of the QuantSeq Kits, the RS-Globin Block solution replaces the RNA Removal Solution 1 (RS1) at step 5. After this, follow the protocol as written, proceeding with the incubation in step 6, and the addition of the RNA Removal Solution 2 (RS2) in step 7.

Currently Globin Block Modules are available for human (Homo sapiens) and pig (Sus scrofa). These contain species-specific globin blockers. The cross-reactivity of these solutions for globin reduction in other species has not been evaluated. If you are interested in globin block solutions for additional species please contact us at support@lexogen.com and tell us which species you are interested in.

Yes, the Globin Block Modules can also be used to prepare QuantSeq libraries from low quality total RNA input from blood (RIN <6). This will also result in a reduction of reads mapping to globin genes.

Input amounts down to 5 ng have also been tested and can be used with adjustments to PCR cycle numbers. For further information please contact support@lexogen.com.

We strongly recommend performing a qPCR assay to determine the optimal number of PCR cycles for the library amplification. Information on the qPCR assay can be found in the QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina User Guide (Appendix, E). The PCR Add-on Kit for Illumina (Cat. No. 020.96) includes PCR and Enzyme Mix reagents and P7 primer (7000) that are required for the qPCR assay. In addition, SYBR Green I Nucleic Acid Gel Stain (10,000X in DMSO, Sigma-Aldrich Cat. No. S9430) is also required (user provided).

ATTENTION: The use of SYBR Green qPCR Mastermixes is not recommended and may result in incorrect cycle number calculation.

Typically, libraries prepared from blood total RNA using Globin Block require one additional PCR cycle compared to libraries made with the standard QuantSeq Kit reagents.

Libraries prepared from human blood RNA with standard QuantSeq Library Prep Kit and protocol display distinct peaks in bioanalyzer traces at 197 bp, 212 bp, 222 bp, 235 bp, and 312 bp, which correspond to abundant globin mRNA library fragments (HBA1, HBA2, and HBB). These peaks are reduced in libraries prepared with the RS-Globin Block, Homo sapiens module (RS-GBHs), as shown below in Figure 1 (50 ng whole blood total RNA).

Globin Block libraries can also display: a longer library size distribution, and/or, peaks present at larger molecular weights (~400 bp and higher).
FAQ_Figure1_GlobinBlock

Figure 1 | Bioanalyzer traces for human QuantSeq FWD libraries prepared with (+Globin Block) and without (Standard) the RS-Globin Block solution (RS-GBHs, Cat. No. 070.96). Replicate libraries were prepared from 50 ng of whole blood (WB) RNA with the Standard QuantSeq FWD protocol (blue and red traces), versus QuantSeq +Globin Block (green and turquoise traces). RNA was isolated using the SPLIT RNA Extraction Kit without red blood cell lysis (Lexogen). Grey arrows indicate major globin peaks reduced in +Globin Block Libraries.

Pig blood libraries prepared with RS-Globin Block, Sus scrofa (RS-GBSs) also show an altered peak profile compared to libraries prepared with standard QuantSeq RS (see Figure 2 below).

FAQ_Figure2_GlobinBlock
Figure 2 | Bioanalyzer traces for pig QuantSeq FWD libraries prepared with (+Globin Block) and without (Standard) the RS-Globin Block solution (RS-GBSs, Cat. No. 071.96). Replicate libraries were prepared from 100 ng of whole blood (WB) RNA with the Standard QuantSeq FWD protocol (blue and red traces), versus QuantSeq +Globin Block (green and turquoise traces). RNA was isolated using the Preserved Blood RNA Purification Kit + Dnase I Kit (Norgen Biotek). Grey arrows indicate major globin peaks reduced in +Globin Block Libraries.

Globin reduction is best evaluated prior to sequencing by preparing control libraries from the same RNA sample using the standard QuantSeq protocol, alongside libraries prepared with QuantSeq and RS-Globin Block.

The percentage of reads mapping to globin mRNAs is calculated as the fraction of read counts uniquely mapping to globin genes divided by the total number of uniquely mapped reads.

Uniquely mapped read counts are found in the read_counts.txt files given as output from the QuantSeq Data Analysis Pipeline on the Bluebee® Genomics Platform.

Lists of Ensembl gene IDs for human and pig globin genes are provided in the tables below.

For further information on data analysis for QuantSeq libraries prepared with Globin Block Modules please contact support@lexogen.com.

Table 1. Ensembl Gene IDs for human globin genes.

Gene Symbol Ensembl Gene ID
HBQ1 ENSG00000086506
HBZ ENSG00000130656
HBA2 ENSG00000188536
HBG2 ENSG00000196565
HBA1 ENSG00000206172
HBM ENSG00000206177
HBZP1 ENSG00000206178
HBE1 ENSG00000213931
HBG1 ENSG00000213934
HBD ENSG00000223609
HBBP1 ENSG00000229988
HBB ENSG00000244734

Table 2. Ensembl Gene IDs for pig (Sus scrofa) globin genes.

Gene Symbol Ensembl Gene ID
HBZ ENSSSCG00000007975
HBM ENSSSCG00000007977
HBE1 ENSSSCG00000014726
HBB ENSSSCG00000014725
HBA ENSSSCG00000007978

Unique Molecular Identifiers (UMIs) are short random nucleotide sequences that are added during library generation (prior to PCR). UMIs act as tags that allow the accurate identification of PCR duplicates in sequencing data. PCR duplicates can arise during library amplification if too many cycles are used and can therefore generate a bias in sequencing read counts for downstream analysis of gene expression levels. Removing PCR duplicates can therefore remove any such biases.

When RNA-Seq libraries are prepared from more restricted regions of the transcript, e.g., in amplicon sequencing, and also QuantSeq – which generates a 3’ tag per transcript – there is a higher chance that identical priming occurs on unique transcripts or first strand cDNA molecules, than when using protocols for whole-transcriptome RNA-Seq involving RNA fragmentation. This results in sequencing reads originating from unique transcripts having identical mapping coordinates and sequences. Without UMIs, these reads would be wrongly classified as PCR duplicates and collapsed, resulting in inaccurate read count data. Including UMIs during library generation however clearly distinguishes unique priming events from PCR duplicates, and allows for accurate de-duplication of sequencing reads.

For QuantSeq FWD library prep, UMIs are added during second strand synthesis (for details, see FAQ 4.2 below). With a length of 6 nucleotides, a total of 46, or 4,096 UMIs are possible.

UMIs are most useful for evaluating and removing PCR duplicates in the following cases:

  • Low input amounts (≤10 ng total RNA)
  • FFPE RNA
  • QuantSeq Flex targeted RNA-Seq library prep (can be incorporated in custom primers – See QuantSeq-Flex V2 User Guide, Appendix D, p.28)
  • For very high sequencing depth (e.g., ≥30 M reads per sample. Note the recommended minimum read depth for QuantSeq is 3-4 M reads per sample).

UMIs are likely not required when input amounts are high (≥50 ng). For input amounts of 10 – 50 ng of total RNA, UMIs could be recommended for samples with reduced or variable RNA quality.

Unique Molecular Identifiers (UMIs) can be incorporated into QuantSeq FWD libraries during second strand synthesis. The UMIs are 6 nt long and are positioned 5’ of the random priming sequence in the primers used for second strand synthesis. In addition to the standard QuantSeq FWD Library Prep Kit (Cat. No. 015), the UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1, Cat. No. 081) is required. This module provides the UMI Second Strand Synthesis Mix (USS) that contains the UMI-tagged random primers for second strand synthesis.

The UMI Second Strand Synthesis Module, is used at step 7 of the protocol, instead of the standard Second Strand Synthesis Mix 1 (SS1) from the standard QuantSeq FWD Kit (Cat. No. 015). With this simple solution exchange there are no additional steps added to the QuantSeq library prep protocol. Figure 1 shows how UMIs are added to QuantSeq libraries during second strand synthesis.

UMI-Workflow

Figure 1 | UMIs are added during the second strand synthesis step of the QuantSeq workflow.

Yes, the UMIs are random 6 nt sequences that will be added to all samples. Therefore, the UMI sequence cannot be used to uniquely identify the different samples – only to tag distinct random priming events during library generation. The i7 (and i5) indices are added during library amplification and are still required if multiple libraries need to be uniquely barcoded and pooled for sequencing in a single lane or run. The i7 and i5 index sequences are read-out during sequencing during index reads 1 and 2, respectively and are also required for sample demultiplexing after sequencing. The i7 indices required for the final amplification of QuantSeq libraries are included in every QuantSeq Library Prep Kit. In addition, 96 i5 indices are also available from Lexogen (Cat. No. 047.96) to enable preparation of uniquely dual-indexed libraries for Illumina sequencing.

The UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1) is compatible with the following kits and modules:

  • QuantSeq 3’ mRNA-Seq Library Prep Kits for Illumina (FWD, Cat. No. 015)
  • QuantSeq-Flex Targeted RNA-Seq Library Prep Kit V2 with First Strand Synthesis Module (Cat. No. 033) using oligodT or target-specific first strand synthesis priming and random primed second strand synthesis
  • QuantSeq Flex First Strand Synthesis Module for Illumina (Cat. No. 026)
  • Globin Block Modules for QuantSeq (Human and Pig, Cat. No. 070, 071)
  • I5 Dual Indexing Add-on Kits for QuantSeq/SENSE (Cat. No. 047)
  • PCR Add-on Kit for Illumina (Cat. No. 020)

This UMI module is not compatible with the following kits:

  • QuantSeq 3’ mRNA-Seq Library Prep Kit REV (for Illumina, Cat. No. 016)
  • QuantSeq 3’ mRNA-Seq Library Prep Kit for Ion Torrent (Cat. No. 012)
  • QuantSeq-Flex Targeted RNA-Seq Library Prep Kit V2 with Second Strand Synthesis Module (Cat. No. 034)
  • SENSE mRNA-Seq and SENSE Total RNA-Seq Library Prep Kits (for Illumina: Cat. No. 001, 009; and for Ion Torrent: Cat. No. 006)
  • CORALL Total RNA-Seq Library Prep Kit (Cat. No. 095, 096)
  • Small RNA-Seq Library Prep Kit for Illumina (Cat. No. 052, 058)

The UMI Second Strand Synthesis Module for QuantSeq is also not compatible with, and not recommended for use with any other external library prep protocols or kits.

The UMI module can also be used for preparing QuantSeq-Flex libraries using targeted priming for first strand cDNA synthesis and random priming for second strand synthesis (i.e., using the QuantSeq-Flex Targeted RNA-Seq Library Prep Kit V2 with First Strand Synthesis Module, Cat. No. 033). For use with QuantSeq-Flex, the UMI Second Strand Synthesis Mix (USS) also replaces the Second Strand Synthesis Mix 1 (SS1) from the standard kit at step 7 of the protocol for Random Primed Second Strand Synthesis (QuantSeq-Flex User Guide, 015UG058, p.16).
The UMI sequence (6 nt) and following 4 nt spacer is read-out at the beginning of Read 1. Hence, additional nucleotides at the 5’ end of each read will be taken up by the UMI + spacer. We therefore recommend a minimum read length of 75 bp (i.e., SR75) or longer (up to SR150) for sequencing QuantSeq FWD libraries containing UMIs.

Automated data analysis for QuantSeq FWD-UMI libraries is available on the BlueBee® Genomics Platform (Figure 2).

To analyze data from QuantSeq FWD libraries that contain UMIs, simply use the activation code included with your QuantSeq FWD library prep kit and select the respective “FWD-UMI” pipeline when setting up your data analysis run.
Please note that the number of free data analysis runs available with the activation code included with each QuantSeq FWD kit, is equal to the number of reactions in the kit (e.g. You get 96 data analysis runs with a 96 prep QuantSeq FWD Kit (Cat. No. 015.96), meaning you can analyze sequencing data for 96 .fastq files).

Therefore, you should choose either the FWD or FWD-UMI pipeline, depending on how your libraries were prepared. If you wish to run both options, additional Activation Codes will need to be purchased (Cat. No. 090). If you start the wrong pipeline for your data you can abort or stop the run before it is completed without losing your allocated runs. If the run is completed then additional runs will need to be purchased.

QuantSeq FWD libraries prepared from blood using Globin Block (RS-GBHs or RS-GBSs, Cat. No. 070 and 071) should be analysed using the standard FWD pipeline for data analysis; unless UMIs are also included and then the FWD-UMI pipeline should be selected.

Be sure to only select the “FWD-UMI” pipelines for UMI-containing libraries, otherwise duplicate reads will not be collapsed.

NOTE!
Do not run the “FWD-UMI” pipelines for QuantSeq FWD (standard) libraries that do not contain UMIs. This will incorrectly collapse reads and result in incorrect read count data. Use only the FWD pipelines for standard QuantSeq FWD libraries!

Do not use activation codes provided with the QuantSeq REV Kit (Cat. No. 016) for analysis of FWD-UMI libraries. The UMI Second Strand Synthesis Module is not compatible with the QuantSeq REV Kit.

ATTENTION: Data Analysis access is only free using the provided code, for fastq input files up to 1.5 GB in size. Larger fastq file sizes can only be processed using activation codes for large file sizes, which can be purchased additionally (Cat. No. 093 for FWD, or Cat. No. 094 for REV library types, respectively).

For further information on the Data Analysis pipelines available for different species on the BlueBee® Genomics Platform, see QuantSeq FWD FAQ 1.27.

Alternatively, a Linux/Unix-compatible data analysis tool package for QuantSeq FWD-UMI libraries (collapse_UMI_bam) is available from Lexogen upon request. This package contains two analysis tools that can be integrated into existing QuantSeq Data Analysis pipelines (e.g. see Figure 2): umi2index (input: raw fastq.gz file(s)), which trims the UMI (and spacer) from each read and adds the UMI to the read identifier; and collapse_UMI_bam (input: aligned .bam files), which collapses reads that have identical mapping coordinates and UMI sequences to remove duplicates, generating a filtered .bam file that contains de-duplicated, uniquely-mapped reads. For more information on collapsing reads using UMIs please contact support@lexogen.com.

QuantSeq_Bluebee_Pipeline_Graphic
Figure 2 | QuantSeq FWD-UMI Data Analysis Pipeline available on the BlueBee® Genomics Platform.

Lane mix and PhiX concentrations required for sequencing QuantSeq FWD-UMI libraries on NextSeq 500/550 vary slightly from those used for standard QuantSeq FWD libraries. For NextSeq 500/550 runs that consist solely of QuantSeq FWD-UMI libraries, we recommend loading 1.8 pM of the lanemix with 15-30 % PhiX spiked-in. For HiSeq 3000/4000 instruments we recommend using 5 % PhiX spike in (15 % spike-in may lead to PhiX over-clustering). For all other instruments we recommend adding 15-30% PhiX spike-in to the lanemix and following instrument-specific loading guidelines defined by Illumina.

Loading amounts may still need to be adjusted to obtain optimal cluster densities for your respective instrument, or if sequencing chemistries are changed. Inaccuracies in library quantification can affect the loading amount required. Please ensure accurate quantification of lane mixes and lane mix dilutions when preparing for loading.

In general, we do not recommend multiplexing Lexogen libraries with libraries from other vendors in the same sequencing lane (see FAQ 1.30 for further details).

QuantSeq FWD-UMI libraries contain a 4 nt spacer that separates the 6 nt UMI at the start of the read from the random priming sequence that initiates second strand synthesis. The spacer has the same sequence (TATA) in all library fragments which generates low diversity signals for cycles 7-10 of Read 1. This low diversity signature is visible in both sequencing run results (e.g. SAV files, or basespace run results), and FastQC analysis results. The figures below show the FastQC per base sequence content for QuantSeq FWD (standard, no UMI, Figure 3) and QuantSeq FWD-UMI (Figure 4) libraries.

We recommend adding a minimum of 5 – 15 % PhiX (maximum 30%) spike-in when sequencing QuantSeq FWD-UMI libraries on all Illumina instruments. The higher PhiX percentage corrects for the low diversity of the spacer.

ATTENTION: The optimal PhiX spike in percentage may differ between instruments. See the QuantSeq FWD FAQ 1.24 for full loading amount guidelines for different Illumina instrument types.

QS_UMI_4.9_Fig-4_std
Figure 3 | FastQC Per Base Sequence Content profile for QuantSeq FWD library pool sequenced on NextSeq 500 (no UMIs).

QS_UMI_4.9_Fig-3_umi
Figure 4 | FastQC Per Base Sequence Content profile for QuantSeq FWD-UMI library pool sequenced on NextSeq 500.

Please note, the QuantSeq FWD-UMI data analysis pipeline available on the BlueBee® Genomics Platform trims the UMI and spacer from the beginning of Read 1 before read Quality Control is performed. Therefore, FastQC reports for raw and trimmed reads look the same as for Standard QuantSeq FWD libraries (without UMIs, see FAQ 4.7 for pipeline details).

Yes! Free access to QuantSeq FWD-UMI data analysis pipelines is available on the BlueBee® Genomics Platform.

To analyze QuantSeq FWD-UMI data, simply use the activation code included with your QuantSeq FWD kit and select the respective “FWD-UMI” pipeline when setting up your data analysis run on BlueBee® (for further information see FAQ 4.7).

The activation codes are printed on a sticker that is attached to the side of the small reagent box inside the main QuantSeq Kit FWD box (see FAQ 1.28). Activation codes for additional runs can also be purchased from Lexogen via the webshop (Cat. No. 090).

The Lexogen i5 6 nt Unique Dual Indexing Add-on Kit (5001 – 5096) provides a 96-well plate containing 96 unique i5 indices (Cat. No. 047.96). This kit is designed for unique dual indexing in combination with Lexogen’s 96 i7 6 nt indices (included in all QuantSeq 3’, QuantSeq-Flex, CORALL, and SENSE library prep kits (Cat. No. 001, 009, 015, 016, 033, 034, 035, 042, 095, and 096; and separately: Cat. No. 044.96)). Up to 96 uniquely dual-indexed libraries can be prepared for sequencing in a single lane or run.

The Lexogen i5 6 nt Dual Indexing Add-on Kit (5001 – 5004, Cat. No. 047.4) provides four different i5 index primers (5001 – 5004). Each tube contains sufficient volume for preparing 24 (Cat. No. 047.4×24), or 96 (Cat. No. 047.4×96) libraries. This kit is therefore ideal for combinatorial dual indexing strategies (see FAQ 5.6). In combination with 96 i7 indices a maximum of 384 (4 i5 x 96 i7) dual-indexed libraries with different i5 / i7 index combinations can be multiplexed in a single sequencing lane or run.

The Lexogen i5 6 nt Dual Indexing Add-on Kits are compatible with all QuantSeq, CORALL, and SENSE Library Prep Kits for Illumina, specifically:

  • QuantSeq 3’ mRNA-Seq Library Prep Kits (FWD and REV) for Illumina (Cat. No. 015 and 016)
  • QuantSeq-Flex Targeted RNA-Seq Library Prep Kits V2 for Illumina (Cat. No. 033, 034, and 035)
  • SENSE mRNA-Seq Library Prep Kit V2 for Illumina (Cat. No. 001)
  • SENSE Total RNA-Seq Library Prep Kit for Illumina (Cat. No. 009, 042)
  • CORALL Total RNA-Seq Library Prep Kit (Cat. No. 095, 096)

These add-on kits are not compatible with the Small RNA-Seq Library Prep Kit for Illumina (Cat. No. 052, 058). This is because the internal adapters in the small RNA libraries differ from those used in QuantSeq, CORALL, and SENSE libraries.

The Dual PCR Mix (Dual PCR, purple cap) must be used instead of the standard PCR Mix (PCR, yellow cap for QuantSeq, CORALL, and SENSE mRNA-Seq Kits; white cap for SENSE Total RNA-Seq Kits), for amplification of dual-indexed libraries. The Dual PCR Mix contains no primers, which enables the i5 and i7 index primers of your choice to be added to each library, in the selected combinations. If the standard PCR Mix (yellow/white cap) is used together with i5 and i7 index primers, there will be a fraction of insert fragments in the final library that will not contain any i5 index. Therefore, reads will be lost during demultiplexing.

The i5 and i7 indices are added during the final library amplification. Please follow the detailed protocol for Endpoint PCR and Post-PCR Purification steps for dual-indexed library preparation in the Instruction Manual for the Lexogen i5 6 nt Dual Indexing Add-on Kits (047IM109). Differences to the protocol for amplification of single-indexed QuantSeq libraries are as follows:

  • The Dual PCR Mix (purple cap) must be used instead of the standard PCR Mix (yellow/white cap).
  • The total volume of the PCR is 35 μl.
  • The volume of Purification Beads (PB) to add for post-PCR purification is 35 μl (or 31.5 μl for libraries generated from low input or degraded / FFPE RNA samples).

The Dual PCR Mix is provided in the Lexogen i5 6 nt Dual Indexing Add-on Kits along with the i5 index primers. The i7 index primers and Enzyme Mix (E3 – or E2 for SENSE Kits) are provided in the respective library prep kit. The Enzyme Mix (E), from the PCR Add-on Kit for Illumina (Cat. No. 020.96) can also be used for dual indexing endpoint PCR. The purification reagents for Post-PCR Purification are also provided in the standard library prep kits.

The i5 index sequence to enter into Illumina sample sheets must be in forward (i5) or reverse complement (i5rc), depending on the instrument and flow cell type used for the sequencing run. The i5 sequences in both orientations are provided for download in the Lexogen i7 and i5 Index Sequences excel file, available from the QuantSeq, CORALL, and SENSE product pages, and Index Balance Checker Support Tools page.

All i5 and i7 indices are 6 nt long, however if you are running 8 cycle index reads, then the first 2 nucleotides from the adjacent adapter sequence need to be added to the sequence in the sample sheet. These are: AC for i5 (forward orientation), and GT for i5rc (reverse complement orientation).

Dual indexing in general, which uses both i7 (Index 1) and i5 (Index 2) indices, enables multiple RNA-Seq libraries to be multiplexed for a single sequencing lane or run. Using dual indexing, the combination of both i5 and i7 indices determines the unique barcode for sample identification and increases the accuracy of read identification when the sequencing data is demultiplexed.
There are two types of dual indexing: combinatorial dual indexing, and unique dual indexing (Fig. 1).

i5-dual-indexing-strategies
Figure 1 | Comparison of combinatorial and unique dual indexing strategies. Regardless of the strategy used, each library (A – F) must have a unique combination of i5 (purple, 5001-5006) and i7 indices (yellow, 7001 – 7006). Using combinatorial dual indexing the same i5 and i7 indices can be used for different libraries, so long as the other index is unique (e.g., libraries B and C both have i5 index 5002, but different i7 indices (7002 or 7003)). For truly unique dual indexing, each i5 and i7 index can be used only once, for a single library. In this way only a maximum of 96 uniquely dual-indexed libraries can be multiplexed in a single sequencing lane or run.

Combinatorial dual indexing greatly enhances the multiplexing capacity of a sequencing lane or run. Using Lexogen’s 96 i5 x 96 i7 6 nt indices in combination, up to 9,216 libraries can be uniquely barcoded with different i5 / i7 combinations for multiplexed sequencing in a single run or lane. In doing so, the same i7 and i5 indices can be used for different individual libraries in the pool, so long as the i5 / i7 combination is different for each library.

Unique Dual Indexing differs from combinatorial dual indexing, whereby each i5 and i7 index is present only once in the pool of multiplexed libraries. Unique dual indexing is required to detect index hopping and prevent read mis-assignment during demultiplexing. Index hopping can happen when either the i7 or i5 index of a particular library fragment is switched during cluster generation. Index hopping occurs more frequently on patterned flow cell instruments (HiSeq 3000 / 4000 and NovaSeq) that use ExAmp chemistry, and when excess indexing primers, or adapter dimers are present.

Index hopping can also be controlled by repurifying library pools prior to sequencing to remove excess adapter dimers and free index primers. Lexogen’s Purification Module with Magnetic Beads (Cat. No. 022.96) can be used for this repurification step. Briefly, add 0.9 volumes of Purification beads (PB) to the library pool, mix well and incubate for 5 minutes at room temperature, then follow steps 30 onwards in the QuantSeq User Guide Detailed protocol (see 015UG009 for protocol details).

Hamming distance refers to the number of nucleotide exchanges (substitutions) needed to convert one index sequence to another. All Lexogen i5 and i7 indices are designed with a minimum hamming distance of 3, meaning that each i7 index differs from all other i7 indices by at least 3 nucleotides, and each i5 index differs from all other i5 indices by at least 3 nucleotides. For example, considering 7001: CAGCGT and 7003: ACCAGT, the underlined nucleotides 1 – 4 would all need to be changed or mismatched to convert 7001 to 7003, or vice versa. Therefore, the hamming distance between these index sequences is 4.

With a hamming distance of 3, you can detect up to 2 errors (mismatches). However, if you want to perform error correction, only 1 error (or mismatch) can be detected and corrected. We recommend turning off error correction during demultiplexing and allowing for zero mismatches. This results in slightly higher rates of unidentified reads. However, the accuracy of index identification is increased by avoiding misassignment, thus giving you more accurate sequencing reads for each library (see also FAQ 5.6).

The naming of the i5 Dual Indexing Add-on Kits, and the naming of the i5 and i7 Index Sets was recently updated in early 2019 to also specify the barcode length (6 nt). In addition all i5 and i7 indices are now provided in clear 96-well plates. This change is being phased into all newly-produced library prep and Dual Indexing Add-on kits. The i5 and i7 6 nt index sequences and layouts in the 96-well plate formats have not been changed!

New i5 Dual Indexing Add-on Kit Naming:
Lexogen i5 6 nt Dual Indexing Add-on Kit (5001 – 5004; Cat. No. 047.4×24/.4×96)
Lexogen i5 6 nt Unique Dual Indexing Add-on Kit (5001 – 5096; Cat. No. 047.96)

New Index Set Naming:
Lexogen i5 6 nt Index Set (5001 – 5004; Cat. No. 047.4×24/.4×96, provided in tubes)
Lexogen i5 6 nt Index Set (5001 – 5096; Cat. No. 047.96, provided in 96-well plate)
Lexogen i7 6 nt Index Set (7001 – 7096; Cat. No. 044.96, provided in 96-well plate)

The Lexogen i5 6 nt Dual Indexing Add-on Kits contain the indexed i5 adapters only. They can be used to introduce dual indexing for all Lexogen library prep kits that include the Lexogen i7 6 nt Indices (015, 016, 095, 001). The indices of the i7 and i5 6 nt Index Sets are 6 nucleotides long. The Lexogen UDI 12 nt Unique Dual Indexing Kits contain pre-mixed i5 and i7 adapters, each 12 nucleotides long.

When using the UDI 12 nt Sets for introduction of Unique Dual Indexing to libraries prepared with Lexogen kits that include the Lexogen i7 6 nt Indices (015, 016, 095, 001), the PCR (yellow) from the library prep kits has to be replaced by the Dual PCR Mix (purple) from the UDI 12 nt Add-on kits and the 12 nt UDIs are used instead of the i7 6 nt indices.

For convenience the QuantSeq FWD and CORALL kits are available as versions containing the UDI 12 nt Sets instead of the i7 6 nt Index Sets (113-115 and 117-119, respectively).

For more information on Lexogen Unique Dual Indexing solutions please visit www.lexogen.com/indexing/12nt-dual-indexing-kits/.

Lexogen UDI 12 nt Unique Dual Indexing Add-on Kits are compatible with QuantSeq FWD and REV (Cat. No. 015, 016), QuantSeq-Flex (Cat. No. 033, 034, 035), CORALL (Cat. No. 095, 096) and SENSE mRNA V2 (Cat. No. 001) Library prep kits.

The Lexogen UDI 12 nt Unique Dual Indexing Sets are not compatible with Small RNA library preparation kit (Cat. No. 052).

Downloads

pdf  QuantSeq Application Note (Nature Methods, December 2014) – external link
pdf  Application Note for QuantSeq
pdf  Product Flyer – introducing QuantSeq data analysis in Partek Flow
pdf  Application Note for Globin Block Modules
pdf  Application Note for Automation of QuantSeq 3’ mRNA Kit on the Agilent NGS Workstation
pdf  Application Overview Flyer for Automation of QuantSeq 3’ mRNA Kit on Beckman Biomek NGS Workstation

QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina

pdf  User Guide – update 03.04.2020
pdf  User Guide HT – update 07.09.2018
pdf  User Guide – QuantSeq 3’ mRNA-Seq Integrated Data Analysis Pipelines on BlueBee® Genomics Platform
pdf PCR Add-on Kit for Illumina Instruction Manual – update 03.07.2019
pdf Lexogen i5 6 nt Dual Indexing Add-on Kits (5001-5096) Instruction Manual – update 12.03.2019

pdf Lexogen i7 and i5 Index Sequences – update 05.05.2020
pdf Library Quantification Calculation File

autoQuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina

  • Perkin Elmer: Sciclone® / Zephyr®
  • Hamilton: Microlab STAR / STARlet
  • Agilent: NGS Workstation (NGS Bravo Option B)
  • Beckman Coulter: Biomek FXP, and Biomek i7
  • Eppendorf: EpMotion® 5075

Please inquire at info@lexogen.com for the automation scripts

Material Safety Datasheets

pdf  MSDS information for QuantSeq Expression Profiling Library Prep Kits – update 17.12.2019
pdf  MSDS information for Globin Block Modules for QuantSeq – release 11.04.2018

If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.

QuantSeq Bioinformatics Data Analysis

Find more about the QuantSeq Data Analysis here.

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