Library preparation is the first step in sequencing. The aim is to enable DNA or RNA to be identified in the sequencing flowcell. The success of obtaining high-quality RNA sequencing data largely depends on the selection of the best library preparation protocol. No single library preparation method is suitable for all research needs. The study objectives, experimental materials and sequencing parameters determine the choice of library preparation protocol.
How to Choose the Most Suitable Library Preparation Solution?
The purpose of the study is one of the key factors. For example, when studying long non-coding RNA (lncRNA) lacking poly(A) tail length, an approach based on the depletion of the abundant fraction of rRNAs is generally chosen; when gene expression profiles are the main focus of the study, an enrichment strategy based on the enrichment of RNA fragments containing poly(A) tails, which will discard most other RNAs before analysis, is a common choice.
Both approaches to library preparation have their own advantages and areas of applicability. rRNA depletion methods are generally superior to poly(A) enrichment in terms of thoroughness of transcriptome analysis (identifying more RNA classes and unique transcriptome features), with the primary goal of providing an estimate of RNA abundance. Removal of rRNA from the total RNA of the extracted sample consists of (1) a sequence-specific probe that can hybridize with the rRNA. After hybridization with biotin-labeled DNA or locked nucleic acid (LNA) probes, rRNAs are removed using streptavidin magnetic beads; (2) design of antisense DNA oligonucleotides complementary to the rRNA, followed by digestion using RNase H enzyme.
Poly(A) enrichment method has been shown to be superior in comparison to exon alignment rates and clinical sequencing (gene quantification, expression-based patient classification and marker gene expression quantification) are excellent. Enrichment can be performed with anchored oligo(dT) coupled to either magnetic or cellulose beads.
Each single-stranded RNA transcript in a cell is synthesized from one of two DNA strands. Therefore, strand information is essential for the analysis of many studies. During RNA library preparation, RNA transcripts are converted back to complementary DNA (cDNA). Strand-specific RNA library preparation improves RNA-seq by accurately identifying antisense transcripts and non-coding RNAs, and distinguishing the boundaries of closely positioned or overlapping genes. This approach facilitates the detection of novel RNA species and the quantification of RNAs.
In addition to the previously mentioned information, another factor in choosing an RNA library construction protocol is sample quality. Low input sample is common in transcriptome sequencing, including FFPE, blood-derived RNA as well as exosomal RNA. Some tagmentation techniques (in-solution and on-bead, etc.) have been developed to obtain sequence-ready RNA libraries more quickly and easily, reducing the need for sample input and producing libraries with more homogeneous and uniform coverage.
Quality Assessment of RNA Library Constructs
Quality assessment of RNA library construction includes the level of the raw data itself (alignment statistics, quality scores, quantified values, ribosome content, etc.). The second is to compare the results of differential expression analysis (e.g., exons, introns or ligations). The third is the pathway enrichment analysis. The comparative analysis of different methods allows to determine which library preparation or commercial kit is better to use.
References:
Sarantopoulou D, Tang S Y, Ricciotti E, et al. Comparative evaluation of RNA-Seq library preparation methods for strand-specificity and low input. Scientific reports, 2019, 9(1): 1-10.
Jaksik R, Drobna-Śledzińska M, Dawidowska M. RNA-seq library preparation for comprehensive transcriptome analysis in cancer cells: the impact of insert size. Genomics, 2021, 113(6): 4149-4162.
* For Research Use Only. Not for use in diagnostic procedures.
