The NGS Sample Preparation Market in 2026 encompasses a substantial and growing RNA sequencing library preparation segment where the expanding applications of transcriptomics in cancer biomarker research, drug mechanism characterization, clinical diagnostic gene expression profiling, and the emerging spatial transcriptomics field are driving demand for specialized RNA library preparation chemistries and kits that address the specific challenges of RNA — its chemical instability relative to DNA, the dominance of ribosomal RNA requiring depletion strategies, and the diverse RNA molecule types requiring different library preparation approaches for different RNA biology questions.

Ribosomal RNA depletion and poly-A selection represent the two primary strategies for reducing the ribosomal RNA background that constitutes greater than ninety percent of cellular total RNA, with poly-A selection using oligo-dT beads to capture and enrich polyadenylated messenger RNA providing high enrichment efficiency for mature mRNA but losing non-polyadenylated RNA species including non-coding RNAs, circular RNAs, and primary transcripts, while ribosomal RNA depletion using hybridization probes complementary to rRNA sequences that direct rRNA molecules to magnetic bead removal preserves the full transcriptome representation including non-polyadenylated RNAs at the cost of somewhat lower mRNA enrichment efficiency than poly-A selection. FFPE tissue RNA library preparation requires additional enzymatic repair steps to reverse formalin-induced RNA crosslinks and fragmentation that require specialized FFPE-compatible library preparation kits designed for the degraded, fragmented RNA extracted from archival tissue specimens.

Strand-specific RNA library preparation methods that preserve information about which genomic strand each sequenced RNA molecule was transcribed from are becoming standard for research RNA-seq applications where accurate identification of antisense transcripts, overlapping gene expression, and non-coding RNA orientation requires strand information that non-strand-specific libraries cannot provide. Deoxyuridine triphosphate incorporation followed by uracil DNA glycosylase digestion of the RNA-first strand synthesis product before second strand synthesis is the most commonly used strand specificity approach, achieving greater than ninety-nine percent strand specificity that enables confident strand assignment of ambiguous reads mapping to overlapping transcriptional units.

Small RNA library preparation for microRNA, small interfering RNA, and other small non-coding RNA species requires specialized ligation-based approaches that attach RNA adapters to the five-prime and three-prime ends of size-selected small RNA molecules in the fifteen to forty-five nucleotide range, with size selection through gel excision or bead-based size fractionation removing the abundant transfer RNA and ribosomal RNA fragments that would otherwise dominate small RNA libraries without size selection. The strong ligation bias toward certain small RNA sequences over others in standard small RNA library preparation creates representation distortions that randomized adapter ligation approaches using four-nucleotide random sequences at the adapter ligation junction partially mitigate by reducing the sequence context dependence of ligation efficiency that creates differential small RNA capture rates.

Direct RNA sequencing using Oxford Nanopore technology that threads native RNA molecules through nanopores without requiring reverse transcription or cDNA synthesis is enabling library preparation approaches that preserve RNA base modifications including N6-methyladenosine and pseudouridine that carry functional information erased during reverse transcription, with the current limitation of lower throughput and higher raw error rate compared to Illumina cDNA-based RNA-seq being progressively addressed through improved nanopore chemistry and basecalling algorithm development.

Do you think direct RNA sequencing will eventually replace cDNA-based RNA-seq as the standard transcriptomics approach as nanopore accuracy improves, or will the throughput and cost advantages of Illumina short-read approaches maintain their dominance for high-throughput transcriptomics applications?

FAQ

• What are the key quality metrics used to assess RNA sample and library quality before committing to NGS sequencing runs and what action thresholds are typically applied? RNA sample quality is assessed by RNA integrity number from Bioanalyzer or DV200 percentage of fragments above two hundred nucleotides from TapeStation analysis for FFPE samples, with RIN above seven typically required for standard RNA-seq and DV200 above thirty percent the minimum threshold for FFPE RNA-seq, RNA concentration measured by Qubit fluorometry confirming sufficient input for the intended library preparation protocol, and A260/A280 ratio from spectrophotometry confirming absence of protein contamination, while library quality assessment uses Bioanalyzer or TapeStation size distribution confirming target insert size range, qPCR quantification using adapter-specific primers measuring functional library concentration, and optional sequencing of a small fraction of the library for insert size distribution confirmation and rRNA depletion efficiency verification before committing to full-scale sequencing runs.
• How does RNA library preparation for clinical diagnostic applications including gene expression profiling and RNA fusion detection differ from research RNA-seq library preparation in terms of regulatory and quality requirements? Clinical diagnostic RNA library preparation requires substantially higher analytical validation standards than research protocols including demonstration of reproducible performance across RNA input quantity and integrity ranges encountered in clinical specimens, interlaboratory reproducibility across multiple sites for multi-center diagnostic programs, lot-to-lot performance consistency of commercial library preparation kit reagents demonstrated by the manufacturer with certificate of analysis documentation, reference standard RNA controls with certified expression values or known fusion transcripts enabling quality control monitoring in each clinical preparation batch, and validation of the library preparation's performance characteristics as part of the overall clinical assay validation per CAP/CLIA or CE-IVD requirements that the diagnostic laboratory or kit manufacturer must document before clinical use.

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