Small RNAs are RNA molecules less than 200 nucleotides in size that are important in regulating numerous biological processes. Small RNA sequencing (small RNA-Seq) allows for genome-wide profiling to discover new variants of small and microRNAs and analyze the levels of known small RNAs. Due to the high sensitivity of small RNA-Seq, it can be used to analyze low-input, liquid biopsy samples for disease diagnosis and prognosis. However, certain steps in the small RNA-Seq process, particularly during library preparation, are susceptible to bias. To accurately capture true small RNA profiles, these biases should be minimized as much as possible.

Sources of Bias

High-quality data in small RNA sequencing is dependent on multiple factors, including the optimization of RNA extraction. However, since researchers can often perform extraction in their own laboratories, this discussion will focus on biases introduced during library preparation. 

After successful RNA extraction, library preparation begins with the reverse transcription of small RNA into complementary DNA (cDNA) fragments. During this process, two-adapter ligation is done, where DNA oligo adapters are attached to both the 3’ and 5’ ends of small RNA molecules. This process elongates the small RNA and provides annealing sites for the reverse transcription (RT) primer and subsequent PCR and sequencing primers [1]. 

Significant bias can be introduced during the PCR amplification step, where molecules of different lengths may be amplified with varying efficiencies, leading to skewed representation in analysis. However, the ligation step is the primary source of bias, as different affinities of adapters to target molecules can artificially influence results. This can lead to inaccurate readings of small RNA levels, and adapter dimers may form, consuming sequencing space in place of more relevant fragments [1].

Strategies to Mitigate Biases

Mitigating PCR bias can be achieved by incorporating unique molecular identifiers (UMIs). These are indices added to cDNA fragments before PCR amplification, facilitating the bioinformatic identification of PCR duplicates. The QIAseq miRNA Library Kit is an example of a library preparation kit utilizing the two-adapter method with 12bp UMIs. While effective in correcting PCR bias, this kit still uses two-adapter ligation and is susceptible to ligation bias [1].

The most common way to address ligation bias involve either improving the original two-adapter ligation method or opting for a ligation-free approach:

Improved versions of the original two-adaptor ligation

Ligation of the two adaptors with randomized nucleotides: The ligases that attach the adapters to small RNAs have affinities for certain sequences, so they would combine adapters to specific RNA sequences more readily, leading to some small RNA being overrepresented and others underrepresented. Researchers noticed that adding random oligonucleotides to the adapters reduced this bias significantly, ensuring a more equitable representation of small RNA sequences. The NEXTflex Small RNA-Seq Kit v3 is an example of a library prep kit using adapter randomization [2].

Ligation of one adaptor followed by circularization: This method involves ligation at the 3’ end, followed by circularization of the small RNA molecule once the 3’ adapter is attached. The adapter for the 3’ end is blocked by a phosphate group, which prevents self-circularization. Then once the 3’ adapter is attached, the phosphate group is removed, the whole small RNA-adapter molecule is circularized and reverse transcription is performed to obtain cDNA. This approach effectively mitigates 5’ end bias but retains some 3’ end ligation bias [1]. The RealSeq-Biofluids kit employs this single adapter and circularization method [3].

Ligation-free approaches 

Polyadenylation extension with template switching: This approach substitutes 3’ end adapter ligation with polyadenylation, followed by a specific type of reverse transcription to cDNA, where non-template cytidines are added at the 5’ end. Then a specific oligo is annealed to this stretch of nucleotides and serves as a template for the necessary adapters to be added at the 3’ and 5’ ends. Having no ligation step is a significant advantage, as it helps ensure more accurate small RNA quantification. However, a drawback includes a lack of control over the number of adenosines during polyadenylation, making it challenging to distinguish the original from additional adenosines [1]. Takara’s SMARTer smRNA-Seq Kit is an example of a library prep kit using this polyadenylation and template switching approach [4]. 

Probe-based techniques: These hybrid techniques allow direct manipulation on small or miRNAs within total RNA samples without the need for PCR amplification or reverse transcription to cDNA. In general, oligonucleotide tags designed to target specific small RNAs are allowed to attach to the small RNAs of interest, unattached tags are removed, and then the attached tags are quantified using a particular quantification system. Here the quantification step does not necessarily involve sequencing, especially if the tags (and the attached small RNAs) can be quantified based on their characteristics. For example, NanoString’s nCounter miRNA assay uses color-coded barcodes as tags [5], and the FirePlex miRNA assay uses barcodes with different fluorescent intensities. These are very strong protocols without adapter ligation or PCR bias. However, these assays are limited to known miRNAs and may not be suitable for discovering novel or rare small RNAs [1]. 

How Can Genohub Help?

At Genohub, we know that each research next-generation sequencing project is unique, so we will take the time to understand your specific project goals and help define precise project  specifications for you if you’re uncertain what specifications are relevant. There is no charge for you for this initial consultation or any of our services. Once we have a set of well-defined project specifications we can get you quick and accurate quotes from our NGS partners around the globe, compare all the different quotes for you if needed, and then connect you directly with our partner with the best quote, all using our easy-to-use online platform  We review all quotes to ensure that they meet your project needs and that they specify measurable quality guarantees. If and when you decide to move forward with the project, we will actively supervise and manage it to make sure that all quality and turnaround guarantees are met.

Our small RNA sequencing partners are experts in every step of the NGS process, including extraction, library preparation, and data analysis. They are also well aware of the biases that can occur during small RNA sequencing and can recommend the best library kit for your particular samples and project goals.

Our partners also have experience extracting from many different types of samples, but they can work just as well with total RNA or small RNA samples that you have extracted yourself.

Get started today by letting us know about your small RNA sequencing project here: https://genohub.com/ngs/ .

References

1. Benesova S, Kubista M, Valihrach L. Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis. Diagnostics (Basel). 2021 May 27;11(6):964. doi: 10.3390/diagnostics11060964. PMID: 34071824; PMCID: PMC8229417.
2. Bioo Scientific Corporation. (2016). NEXTflex Small RNA-Seq Kit v3 https://perkinelmer-appliedgenomics.com/wp-content/uploads/marketing/NEXTFLEX/miRNA/5132-05-NEXTflex-Small-RNA-Seq-v3-18-07.pdf
3. Real Seq Biosciences. (2023) RealSeq-Biofluids. Realseqbiosciences. https://www.realseqbiosciences.com/library-prep-products/realseq-biofluids
4. Takara Bio Inc. (2024). A SMARTer approach to small RNA sequencing. Takarabio. https://www.takarabio.com/learning-centers/next-generation-sequencing/technical-notes/epigenetic-sequencing/full-length-small-rna-libraries
5. NanoString Technologies, Inc. (2022). nCounter miRNA Expression Assay Kit. https://nanostring.com/wp-content/uploads/2023/03/PB_MK3354_miRNA_r18.pdf