RNA-Seq Library Construction for Illumina Platform

What is Library in Sequencing?

The construction of DNA and RNA libraries, spanning from the initial fragmentation to PCR amplification, is a pivotal process in molecular biology. A gene library serves as a repository of receptor bacteria harboring DNA fragments from a specific organism. These libraries can be broadly classified into genomic libraries, encompassing all DNA fragments, and cDNA libraries, housing partial DNA fragments.

RNA Library Preparation for Next Generation Sequencing is a helpful article to choose the right RNA library preparation solution, such as probe-based, poly(A) enrichment method and rRNAs depletion.

Illumina Sequencing's ingenious Sequencing by Synthesis (SBS) method ingeniously utilizes fluorescently-labeled dNTPs to vividly capture base composition. Meticulous attention to detail during library construction proves pivotal for subsequent sequencing and data interpretation. Particularly in RNA sequencing, the predominant method for gene expression analysis, many opt for strand-specific RNA sequencing, albeit a more intricate process. In this context, we present an overview of Illumina's time-tested RNA library construction protocol.

Overview of Strand-Specific RNA-Seq Library may be a useful article to learn more about stranded RNA sequencing and library construction.

What is An Adapter?

An adapter is essentially a short sequence of bases that acts as a connector between the sequenced DNA fragments and the flow cell. Using the Illumina sequencing platform as an example, it comprises three components: P5/P7, which match or complement the oligonucleotide strands on the flow cell; Read1/2, serving as the binding site for sequencing primers; and the Index, utilized for distinguishing between different samples. The Index functions as the "identity card" for samples within a mixed sample pool, typically consisting of 6nt or 8nt (now commonly 8-10 nt), where different combinations of the four bases generate unique Index identifiers.

There are two primary ways to classify adapters. Based on the position of the Index, adapters can be categorized into single-end Index adapters and double-end Index adapters. Single-end Index adapters feature the Index at one end, usually at P7, while double-end Index adapters incorporate the Index at both P5 and P7. As sequencer throughput increases, double-end Index adapters accommodate more samples compared to single-end ones, hence their widespread adoption.

Illumina predominantly employs a Y-shaped adapter structure.

Library preparation (A) Sequence of adapters (single index) used in our protocol. (Greulich et al., 2021)

What is RNA Library Construction?

Library construction involves the transformation of target RNA or DNA into a format compatible with sequencing instruments. The starting material, extracted from tissues or cells, undergoes fragmentation through mechanical or enzymatic means. In the case of RNA samples, reverse transcription converts RNA into cDNA. Subsequently, the generated DNA is ligated into a junction, and the resulting library undergoes PCR amplification to magnify the intended target fragment. The amplified library is then ready for sequencing on the designated machine.

Library construction strategy. (Kobori et al., 2015)

RNA Library Construction for Illumina Platform

Illumina platform RNA library construction encompasses various RNA libraries, such as mRNA libraries and lncRNA libraries, each tailored to specific RNA types.

Strand-specific RNA-seq preserves the orientation details of transcripts, enabling the distinction between reads originating from the positive or negative strand. This feature enhances the accuracy of gene quantification and the detection of alternative splicing events, proving particularly advantageous for extracting information from natural antisense lncRNAs. It facilitates detailed analysis of gene structure and functionality, thereby enriching our understanding of complex biological processes.

The standard RNA library construction involves the following key processes:

• RNA Enrichment

In both eukaryotic and prokaryotic organisms, ribosomal RNA (rRNA) constitutes up to 80% of total RNA. Sequencing total RNA directly would yield predominantly rRNA-related data. To address this, RNA enrichment methods are employed, including oligo-dT enrichment-based mRNA methods and rRNA removal methods.

RNA Sequencing 101: Poly(A) Tail may be a helpful article for you.

• Oligo-dT Enrichment: Exploiting the poly(A) structural feature at the 3' end of eukaryotic mRNA, oligo(dT) magnetic beads selectively capture all transcribed mRNAs for transcript analysis. This method suits high-quality RNA samples.
• rRNA Removal: This method, applicable to both high and low-quality RNA samples, as well as prokaryotic samples, involves specific oligonucleotide probes binding to rRNA. Subsequent RNase H digestion degrades the probe-bound rRNA, and DNase I is used to remove the DNA probe.

• RNA Fragmentation

Large RNA segments are fragmented into smaller fragments in the presence of divalent cations and elevated temperatures.

• cDNA One-Strand Synthesis

Target RNA is reverse transcribed into the first strand of cDNA. The use of RNase Inhibitor during reverse transcription protects RNA from degradation by RNases. Reverse Transcriptase synthesizes a single strand of DNA complementary to the RNA template in the 5'→3' direction.

• cDNA Second-Strand Synthesis

Single-stranded cDNA synthesized in the previous step is unstable and requires immediate synthesis of the second strand by DNA polymerase I. RNase H removes the RNA strand from the RNA-DNA heteroduplex, and DNA polymerase I, with 5'→3' DNA polymerase activity, catalyzes the synthesis of the second cDNA strand.

These meticulous steps ensure the construction of high-quality Illumina RNA libraries, ready for subsequent sequencing and analysis.

Small RNA Library Construction

Small RNAs, a subset of non-coding ribonucleic acids (ncRNAs), play crucial roles in gene regulation, encompassing miRNA, piRNA, tRF&tiRNA, tRNA, snRNA, and snoRNA. Due to their petite size—such as miRNA (17-25 nt), piRNA (24-35 nt), and tRNA (60-95 nt)—they pose challenges for cDNA synthesis using random primers, as smaller fragments offer fewer binding sites for these primers.

Furthermore, small RNAs lack Poly(A) tails, rendering them unsuitable for synthesis using Oligo dT primers. Consequently, constructing libraries from small RNA samples typically involves RNA ligation, which joins the junctions at the 3' and 5' ends of the RNAs, thereby enabling the construction of strand-specific libraries.

Recommended reading: Small RNA Library Preparation.

FAQ: RNA Sequencing Library Preparation

• • Can libraries be used across different sequencing platforms?

    • Both Illumina and UW sequencing platforms offer distinct advantages and enjoy widespread usage. However, due to the high value of the sequencers and the complexity of the sequencing process, libraries cannot be directly transferred between platforms. Nonetheless, commercially available universal library conversion kits offer a solution by efficiently converting Illumina platform libraries into single-stranded circular libraries compatible with the MGI platform. This conversion typically entails an additional 5-10 cycles of PCR amplification, introducing a degree of redundancy to the library prior to sequencing.

• • How to address the issue of PCR amplification bias?

    • PCR bias is an unavoidable challenge. Fragments with high GC content are less likely to be amplified, which, compounded by the exponential nature of amplification, can affect the detection of certain variants, particularly low-frequency ones. When variant frequencies dip below 1%, authentic variants risk being obscured by background noise introduced during sample handling, nucleic acid library amplification, and NGS. Yet, detecting low-frequency variants holds significant clinical value. For instance, ctDNA analysis proves invaluable in molecular pathology diagnosis and real-time tumor recurrence monitoring. However, ctDNA exhibits a lower positive detection rate compared to live tissue, with the inadequacy of low-frequency detection being a major hindrance.

      So, how can this challenge be overcome? The Unique Molecular Identifier (UMI) offers a proven solution. By incorporating specific UMI sequences at both ends of the amplicon, the original DNA library is tagged at each end. Upon sequencing, not only is the sequence information of the target fragment acquired, but also the UMI information from both ends simultaneously. Employing comparative and consensus family analyses enables effective exclusion of pseudo-mutations stemming from DNA damage, PCR amplification errors, sequencing errors, and other sources of noise.

      Recommended reading: Unique Molecular Identifiers (UMIs): Enhancing Accuracy and Precision in RNA Sequencing.

• • What is the difference between rRNA removal and poly A enrichment in sequencing workflows?

    • In sequencing workflows, both rRNA removal and poly A enrichment are frequently utilized techniques. The key distinction lies in their target molecules: poly A enrichment primarily isolates processed mature mRNAs with poly A tails, whereas rRNA removal extends its scope to include non-mRNAs, such as lncRNAs and introns, along with mRNA precursors, thereby offering a more comprehensive view of the transcriptome.

      Recommended reading: Overview of rRNA Depletion in RNA Sequencing.

References:

1. Kobori, Shungo, et al. "High-throughput assay and engineering of self-cleaving ribozymes by sequencing." Nucleic acids research 43.13 (2015): e85-e85.
2. Greulich, Franziska, et al. "Protocol for using heterologous spike-ins to normalize for technical variation in chromatin immunoprecipitation." STAR protocols 2.3 (2021): 100609.