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.

The QIAseq Targeted RNA Panels have been developed as a Sample to Insight solution for quantitative gene expression profiling using RNA-seq. 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.

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 RNA-seq. QIAseq digital RNA sequencing method (A; molecular barcode counts) showed accurate quantification of all three genes corresponding to different RNA input, whereas traditional targeted RNA-seq (B; read counts) revealed PCR duplication limitation and yielded inaccurate quantification.

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.

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.

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.