Molecular barcodes remove PCR and library construction bias
Start with only 25 ng of total RNA
Simple 1 day library construction workflow
Use with any illumina or Thermo-Fisher NGS instrument
QIAseq Targeted RNA Panels have been developed as a Sample to Insight solution for quantitative gene expression profiling using RNAseq. These panels integrate molecular barcode technology and a two-stage PCR-based library preparation to deliver unbiased and accurate quantification for your digital RNA sequencing results. QIAseq Targeted Panels are wet-bench verified to ensure the highest quality of results.
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Kit containing wet bench-verified assays and reagents for first strand synthesis, molecular barcoding, gene-specific amplification and library preparation for targeted RNA sequencing
Kit containing wet bench-verified assays and reagents for first strand synthesis, molecular barcoding, gene-specific amplification and library preparation for targeted RNA sequencing
Digital sequencing (molecular barcodes) principle
QIAseq Targeted RNA Panels use a digital sequencing method, whereby a unique 12-base random molecular barcode incorporated into the gene-specific primers (GSP1) is used in the first extension step (after mRNA is converted to cDNA). Thus, every extension event yields a unique combination of molecular barcode and target sequence. At the end of sequencing, the relative amount of each mRNA target is determined by the number of unique molecular barcode-target combinations that were sequenced, thereby eliminating PCR duplicates and amplification bias, resulting in more accurate, unbiased gene expression analysis.
One solution to overcome the challenges of gene expression profiling
The QIAseq Targeted RNA Panels have been developed as a Sample to Insight solution for quantitative gene expression profiling using RNAseq. The panels use molecular barcodes and a two-stage PCR-based integrated library preparation to overcome the challenge of PCR duplicates and amplification bias to deliver unbiased, accurate and reproducible gene expression results.
Unbiased and accurate gene quantification (A)
Different amounts (20 ng, 5 ng or 1.25 ng) of universal reference RNA were used to determine expression levels of three genes (BNIP1, CTNND2 and DAPK1) using targeted RNAseq. QIAseq digital RNA sequencing method (A; molecular barcode counts) showed accurate quantification of all three genes corresponding to different RNA input, whereas traditional targeted RNAseq (B; read counts) revealed PCR duplication limitation and yielded inaccurate quantification.
Unbiased and accurate gene quantification (B)
Different amounts (20 ng, 5 ng or 1.25 ng) of universal reference RNA were used to determine expression levels of three genes (BNIP1, CTNND2 and DAPK1) using targeted RNAseq. QIAseq digital RNA sequencing method (A; molecular barcode counts) showed accurate quantification of all three genes corresponding to different RNA input, whereas traditional targeted RNAseq (B; read counts) revealed PCR duplication limitation and yielded inaccurate quantification.
Proprietary primer design delivers gene-specific amplicons (>97% specificity)
Sequencing libraries were prepared using 1.25, 5 or 20 ng universal reference RNA and QIAseq Targeted RNA Panels, ranging from 12-plex to 1000-plex. Sequencing was performed on the Illumina MiSeq, dedicating 1 million reads per sample. Specificity is calculated as percent of trimmed and mapped reads that map to intended targets.
Unmatched uniformity (>97% of assays are within 20% of median molecular tag counts)
A 917-plex gene panel was used to prepare a library from 10 ng of NA12878 reference DNA. All assays were designed to be intra-exon, and are thus single copy on genomic DNA. This allows an estimation of the uniformity of amplicon performance in the library preparation step (e.g., every unique tag equals one captured copy). In terms of raw assay performance, 97.5% of assays are within 20% of mean/median molecular tag counts. For cataloged panels, any assay below 20% is redesigned and replaced. Molecular barcodes entirely remove this variation in RNAseq counting.
Simple procedure
Starting with 25 ng of total unfragmented RNA, cDNA is synthesized, and each cDNA molecule is tagged with a unique molecular barcode before any amplification. The uniquely tagged cDNA molecules then undergo a two-stage PCR step for enrichment and library construction. It takes only 6 hours from RNA sample to targeted library ready for sequencing.
Positive results with as little as 0.2 copies of RNA per cell
ERCC standards, at 86 to 705,500 copies, spiked into universal reference RNA sample and enriched using 384-plex QIASeq Targeted RNA Panel in three technical replicates. (A) Sensitivity measurement. Under standard conditions (20 ng RNA input, 0.5 million MiSeq reads), ≥~100 copies of ERCC transcripts were reliability detected, which is the equivalent of ~0.2 copies per cell. (B) Precision measurement. At >10 barcodes/gene, CV was less than 5% for all targets, indicating high technical reproducibility. This corresponds to ~100 copies target RNA in the sample.
High concordance with qPCR
Expression levels for 384 genes were determined by both QIAseq Targeted RNA Panel and qPCR for Human Brain Reference RNA (HBRR) and Universal Human Reference RNA (UHRR) samples. The expression levels determined by qPCR were normalized to the average of four housekeeping genes (ACTB, B2M, GAPDH and RPLP0) and fold change between the samples was calculated (HBRR/UHRR) for each gene. For QIAseq, the number of unique molecular barcodes per gene were counted and normalized to average number of molecular barcodes for the four housekeeping genes for each sample. The fold change for each gene was then calculated. The QIAseq Targeted RNA Panel and qPCR assays exhibit similar fold-changes in gene expression, highlighting the accuracy of QIAseq-results.
Performance
Accuracy: Innovative digital sequencing (molecular barcode counting) eliminates PCR duplication and amplification bias to deliver the most accurate results (see figure Unbiased and accurate gene quantification) .
Uniformity: The QIAseq Targeted RNA Panel workflow has been optimized to deliver highly uniform sequencing results, to ensure sequencing capacity is utilized very efficiently. In fact, digital sequencing (molecular barcodes) entirely remove this variation in RNAseq counting (see figure Unmatched uniformity – 97% of assays are within 20% of median molecular tag counts).
Reproducibility: The QIASeq Targeted RNA Panel system demonstrates strong correlations across technical replicates, product lots and instruments with correlation coefficients averaging above >0.99, to ensure reliable detection of differences in expression between biological samples
Flexibility: QIAseq panels combine the power of NGS with the accuracy of qPCR to allow multiplexing of several samples per NGS while delivering cost-effective results (see figure Simple procedure).
Principle
Traditional RNA sequencing methods suffer from PCR duplication and amplification bias, resulting in inaccurate gene expression analysis. By introducing molecular barcodes before any amplification takes place, QIAseq Targeted RNA Panels are able to eliminate this issue to deliver accurate and digital quantification of genes (see figure Unbiased and accurate gene quantification).
A unique feature of the QIAseq Targeted RNA Panels is the set of built-in control assays. The gDNA assays control for any gDNA contamination in the RNA sample to ensure reproducible results. The housekeeping gene (HKG) assays are used to normalize data, thereby making sample-to-sample and run-to-run comparisons possible.
Procedure
The QIAseq Targeted RNA Panels workflow begins with converting total RNA into cDNA (see figure Simple procedure). The workflow requires minimal RNA input: as little as 25 ng total RNA can be used. No enrichment or depletion steps are necessary. The molecular barcoding step makes use of molecularly barcoded gene-specific primer (GSP1) in a multiplex primer panel (targeting 12-1000 genes) and an input of 20ng of cDNA equivalent (cDNA made from 20 ng of total RNA). After the barcoding step, the uniquely tagged cDNA is purified over beads to remove residual primers, and a PCR is set up with a second pool of gene-specific adapter primers (GSP2) and the RS2 primer, which primes off of a common tag on the GSP1 primers. This reaction insures that intended targets are enriched sufficiently to be represented in the final library. The number of cycles is kept to a minimum to keep PCR-induced variations in amplification to a low level (any variations are easily corrected and accounted for with the molecular barcodes). Another quick cleanup with beads is performed, and a universal PCR is run with RS2 and FS2 primers, which also adds sample-indexing barcodes to each sample. A final cleanup with beads is performed and the library is complete, and ready for quantification and sequencing.
An integral component of the QIAseq Targeted RNA Panels is data analysis and insight. Data analysis modules have been developed that are comprehensive, yet easy to use. Using these modules require no bioinformatics expertise. Starting with raw reads directly off the sequencer, the QIAseq targeted RNA data analysis tools at QIAGEN’s GeneGlobe portal, provide you with gene counts and fold changes, as well as links for pathway analysis.
Applications
Gene expression profiling
Biomarker research
Confirmation of whole transcriptome sequencing data
