Description
QuantSeq-Pool Sample-Barcoded 3′ mRNA-Seq Library Prep Kit for Illumina
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This product can be used for SARS-CoV-2 research. |
QuantSeq-Pool is the optimal solution for gene expression profiling for large screening projects using sample barcoding, early pooling, and batch processing of up to 96 samples in one reaction providing a workflow that is easily scalable for multiplexing up to 36,864 samples.
QuantSeq-Pool is the newest addition to the QuantSeq product line combining the benefits of the well-established QuantSeq methodology with sample barcoding and early pooling. Due to the convenient workflow and scalable multiplexing capacities, QuantSeq-Pool is the optimal choice for completing gene expression profiling studies in the most cost- and time-efficient way. Not sure whether QuantSeq-Pool is the right version of QuantSeq for you? Please check the QuantSeq selection guide to find out (Fig. 1).
Figure 1 | QuantSeq kit selection guide.
Increased Consistency and Reduced Technical Variability
QuantSeq-Pool 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. Sample-barcodes and Unique Molecular Identifiers (UMIs) are introduced in the first step and are accessible in Read 2 (see workflow). Early pooling and batch processing reduce technical variation in the complete workflow and allow consistent processing of up to 96 samples in parallel. Excellent reproducibility between replicates is shown in Fig. 2.
Figure 2 | Excellent correlation between replicates for individual samples in QuantSeq-Pool. Libraries were prepared from 10 ng UHRR, sequenced, and correlated on gene level. R² was calculated by orthogonal regression.
Fastest Turnaround for Large-scale Projects
Flexibility of Throughput
High Strand-Specificity
Robust Gene Detection at Low Sequencing Depth
QuantSeq-Pool reliably detects 7,500 to 9,000 highly expressed genes at very shallow read depths of 100 K to 1 M reads per sample (Fig. 3).
Figure 3 | QuantSeq-Pool enables robust and consistent gene detection already at low sequencing depth. Libraries were generated from 8 x 10 ng UHRR, sequenced, and gene detection was analyzed for 100 K, 0.5 M and 1 M reads / sample. Number of detected genes was counted at a threshold of > 10 Counts Per Million (CPM).
Cost Saving Multiplexing
QuantSeq-Pool libraries are intended for a high degree of multiplexing: 96 sample-barcodes (i1 indices) are included in the Kit (Cat. No. 139). Additional i5 and i7 indices are required for multiplexing more 96 samples for sequencing. Lexogen UDI 12 nt Sets with pre-mixed i5 and i7 indices offer superior error correction capacity and are recommended for massive multiplexing (Cat. No. 101 – 105, 156). Lexogen’s indices are provided in a convenient 96-well format for multiplexing of up to 9,216 samples per lane for each 96-well index plate. Lexogen UDI 12 nt Unique Dual Indexing Sets are available for up to 384 12 nt UDIs increasing the multiplexing capacity to 36,864 samples per sequencing lane. For further indexing options, please contact support@lexogen.com.
Lexogen also offers 6 nt single indexing sets for i7 (Cat. No. 044.96) and i5 (Cat. No. 047.96). These 6 nt Index Sets can be used separately as single indexes for either i5 or i7, or can be combined for dual indexing.
This high level of multiplexing allows saving costs as the length restriction in QuantSeq saves sequencing space.
Direct Counting for Gene Expression Quantification
Simple Bioinformatics Analysis
Unique Molecular Identifiers
Workflow
or rRNA depletion is needed. For balanced read distribution
in the sequencing experiment the RNA input amount for reverse
transcription needs to be adjusted and normalized across all
samples that shall be pooled.
already contains a partial Illumina-specific Read 2 linker sequence,
Unique Molecular Identifiers (UMIs), and an i1 sample-barcode
to uniquely label each sample.
by pooling. The pool is purified to decrease the volume for
subsequent steps. All further reactions are carried out in batch
on the combined samples to save time and effort.
all samples simultaneously.
and a DNA polymerase. The random primer contains
the Illumina-specific Read 1 linker sequence.
bead-based purification step.
cluster generation are introduced. Optionally, i5 and i7 indices
can be introduced. These additional indices are required
for multiplexing more than 96 samples.
96 additional i5 / i7 Unique Dual Indices (UDIs) are used or
up to 36,864 samples when 384 UDIs are used.
of up to 96 samples which directly correspond to the final
lane pool for a 96-plex sequencing experiment.
correspond to the mRNA sequence. The length distribution of
QuantSeq-Pool libraries supports all sequencing lengths.
Read 2 is required to read out the sequence information for
the UMI and the i1 sample-barcode. Therefore, at least a
limited Read 2 with a read length between 18 and 22 nucleotides
is mandatory for sequencing QuantSeq-Pool libraries.
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.
In both standard QuantSeq and QuantSeq-Pool library generation is initiated by oligo(dT) priming, where the primer contains a partial Illumina-compatible linker sequence. However, in QuantSeq-Pool this primer also contains Unique Molecular Identifiers (UMIs) and an i1 sample-barcode. This early barcoding allows the samples to be combined by pooling after first strand synthesis. All subsequent steps of the protocol can thus be performed on pools containing up to 96 samples. Batch processing significantly shortens the overall library generation time and reduces technical variability between samples within one pool.
Formalin-Fixed, Paraffin-Embedded (FFPE) material is very heterogeneous and highly variable in terms of quality, degree of cross-linking, and accessibility of the mRNA. Therefore, processing with QuantSeq-Pool may lead to uneven read distribution between samples in sequencing experiments. For best practice, the QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina (Cat. No. 015) is recommended for processing of FFPE samples as it allows quality control and quantification of individual libraries. This offers the possibility to re-adjust individual samples for equimolar lane pooling and balanced read distribution during sequencing.
Lexogen’s QuantSeq-Pool 3’ mRNA library preparation protocol is designed to generate sequencing Illumina-compatible libraries from total 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.
No, QuantSeq-Pool requires asymmetric paired-end sequencing, where Read 1 is generated towards the poly(A) tail and directly corresponds to the mRNA sequence. Read 2 will contain the Unique Molecular Identifier (UMI) followed by the i1 sample-barcode. Therefore, it is necessary to sequence Read 2 for sample identification, i1 barcode demultiplexing and UMI deduplication.
The kit includes a set of 96 i1 indices (i1 12 nt RT-Set for QuantSeq-Pool, dried-in sample barcodes in 96-well plate format), which allow up to 96 samples to be sequenced per lane on an Illumina flow cell.
For multiplexing of more than 96 samples, unique dual indexing for the individual pools (containing up to 96 sample-barcoded libraries) is recommended.
Lexogen offers various indexing systems that are fully compatible. Unique Dual Indices (UDIs) with pre-mixed i5 and i7 indices are available in a convenient 96-well format. UDIs are 12 nucleotides in length and provide superior error correction capability for massive multiplexing. Lexogen UDI 12 nt Unique Dual Indexing Sets are available in sets of 96 (Cat. No. 105) for multiplexing of up to 9,216 samples per set, or 36,864 samples with just 384 UDIs.
Lexogen also offers 6 nt single indexing sets for i7 (Cat. No. 044.96) and i5 (Cat. No. 047.96). These 6 nt Index Sets can be used separately as single indices for either i5 or i7, or can be combined for dual indexing.
No, currently the QuantSeq data analysis pipelines on BlueBee are only compatible with standard QuantSeq FWD and REV. QuantSeq-Pool libraries contain the Read 1 linker sequence in the 5’ part of the second strand synthesis primer, hence NGS reads are generated towards the poly(A) tail. Following Demultiplexing, all further steps for Trimming, Mapping, UMI processing, and Counting can be integrated in standard data analysis pipelines. Please contact support@lexogen.com for further information on how to analyze your QuantSeq-Pool data.
The i1 demultiplexing tool is available in a command-line format using the Python programming language. Please contact support@lexogen.com for further information on how to analyze your QuantSeq-Pool data.
No, QuantSeq-Pool uses optimized reagents for pooled processing that differ from QuantSeq reagents. For blood samples, the QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina (Cat. No. 015) is recommended for use with Globin Block Modules. Alternatively, globin mRNA can be removed from blood samples prior to extraction, e.g., by using SPLIT for Blood (Cat. No. 099). RNA extracted with SPLIT for Blood, or similar extraction procedures is fully compatible with QuantSeq-Pool.
Downloads
QuantSeq-Pool Sample-Barcoded 3′ mRNA-Seq Library Prep Kit for Illumina
User Guide – update 18.02.2021
Send us your publication & get the RNA T-shirt!
Product Flyer – release 19.08.2020
i1 12 nt RT-Set for QuantSeq-Pool Sample-Barcode Sequences for Illumina
QuantSeq-Pool Calculator
Material Safety Datasheets
MSDS Information can be found in the Documents page.
If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.
Data Analysis
QuantSeq-Pool libraries contain the Read 1 linker sequence in the 5’ part of the second strand synthesis primer, hence NGS reads are generated towards the poly(A) tail. Demultiplexing of i5 / i7 indices can be carried out by the standard Illumina pipeline. Lexogen i7 and i5 index sequences for 6 nt and 12 nt index systems are available for download at www.lexogen.com. The i1 sample-barcode is located at the beginning of Read 2 and preceded by a 10 nt UMI sequence. Therefore, the i1 barcode is contained within bases at position 11 – 22 of Read 2, depending on the chosen index read out length of 8, 10 or 12 nucleotides. Demultiplexing of i1 sample-barcoded libraries can be performed using Lexogen’s Demultiplexing Tool. All further steps for Trimming, Mapping, UMI processing, and Counting can be integrated in standard data analysis pipelines. For further questions on QuantSeq-Pool data analysis, please contact support@lexogen.com.
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