Description
QIAseq UPXome RNA Library Kits enable single-day, high-throughput next-generation sequencing (NGS) RNA-seq library construction for use with Illumina NGS instruments. These RNA library kits feature ultraplex (UPX) pooling of cDNA and support complete transcriptome or 3’ RNA-seq starting with total RNA or enriched mRNA. When used with the QIAseq UX Index IL UDI Index Kits (sold separately), up to 18,432 samples can be ultraplexed together on a single flow cell. The complete solution includes online access to the RNA-seq analysis portal (for supported species), which allows researchers to start with FASTQ files and perform read alignment, differential gene expression and pathway analysis. Furthermore, qPCR and digital PCR assays for biological verification can be easily identified in QIAGEN GeneGlobe following data analysis. QIAseq UPXome RNA Library Kits are also supported with on-site software through QIAGEN’s CLC Genomics Workbench (sold separately).
QIAseq UPXome RNA Library Kits can be used to make NGS libraries for complete transcriptome or 3’ RNA-seq starting with total RNA or enriched mRNA depending on the workflow selected and type of RNA used. This provides researchers the ability to choose the appropriate library type based on the input RNA, NGS sequencing instrument, read budget and overall project goals. RNA-seq libraries have been successfully constructed from high-quality total RNA, fragmented FFPE RNA and enriched mRNA. To see demo data, login to the RNA-seq analysis portal and go to the QIAseq UPXome RNA Library folder, or download FASTQ files for viewing in your pipeline (Sample Data Library).
QIAseq UPXome RNA Library Kits have several innovative advantages compared to other RNA-library kits: First, the integration of QIAseq FastSelect in the workflow allows for rapid and efficient removal of ribosomal RNA during the fragmentation and cDNA synthesis reaction. In one-step, QIAseq FastSelect removes up to 99% of all unwanted rRNA, even when starting with difficult samples or instances where the RNA is already degraded, such as formalin-fixed paraffin embedded (FFPE) samples. Second, during reverse transcription, each transcript incorporates a unique sample ID. Following the reverse transcription step, 8 to 24 samples of cDNA is pooled (ultraplexed) together and all subsequent library construction steps are performed in a single tube. This simplifies RNA-seq library preparation resulting in a much higher throughput than traditional NGS library preparation methods. This increased productivity can be achieved by simply performing manual library preparations or further magnified through automation of the protocols. During sample indexing and final library amplification, up to 768 different unique dual indices can be utilized. When using the standard protocol, up to 18,432 samples can be sequenced together. (24 samples per pool x 768 unique dual indices).
For transcriptome studies, total RNA is randomly fragmented and QIAseq FastSelect blocks ribosomal and unwanted RNAs. The fragmented RNA is then reverse transcribed to make cDNA, which is labeled with a unique sample ID. For mRNA-seq studies, samples are enriched for mRNA and then randomly fragmented. Next, the mRNA is reverse transcribed to make cDNA, which contains a sample-specific ID. You can choose to create complete transcriptome or 3' RNA-seq libraries by simply modifying the reverse transcription reaction. Following cDNA synthesis, 8 to 24 samples are pooled together into a single library due to the incorporation of the sample-specific IDs. Pooling helps increase library yield for lower input samples. Each library is then amplified and unique dual indices are added using the QIAseq UX Index Kits in a single PCR step. Libraries are quantitated for yield and fragment size, pooled and then run on an Illumina NGS instrument. FASTQ files can be uploaded to QIAGEN GeneGlobe for use with the RNA-seq Analyis Portal for sample demultiplexing, differential gene expression and pathway analysis using commonly used NGS pipelines and algorithms specifically designed for QIAseq UPXome libraries.
