QuantSeq - 3` mRNA-Seq Library Prep Kit FWD for Illumina
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Cost-efficient genome wide analysis of gene expres...
- Cost-efficient genome wide analysis of gene expression
- Saving sequencing depth by generating only one fragment per transcript
- Fast and simple all-in-one protocol: less than 4.5 hours
- Suitable for low input (1 ng total RNA) and FFPE samples
- Free data analysis pipeline for non-bioinformaticians
- UMIs for detection of PCR duplicates available
- NEW! Bundles with up to 384 Unique Dual Indices (UDIs)
QuantSeq FWD contains the Illumina Read 1 linker s...
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 recommended input amount of total RNA is as low as 1 ng. 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.
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.
Deplete globin mRNAs during QuantSeq Library Prep
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.
Deplete the BC1 transcript from mouse brain sample...
Deplete the BC1 transcript from mouse brain samples
New! The BC1 Block Module for QuantSeq prevents the generation of library fragments from the abundant BC1 transcripts that are present in mouse brain samples (Mus musculus, Mm), by blocking their extension during second strand synthesis. The provided RS-BC1 Block solution from the module simply replaces the standard RNA Removal Solution from the QuantSeq Kits (FWD and REV).
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.
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.
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.
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.
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.
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