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Reduced-Bias Small RNA Library Preparation with Gel-Free or Low-Input Options

Next Generation Sequencing (NGS) of small RNAs has significantly improved our understanding of gene regulation, as small RNAs have been shown to fine tune or modulate the expression of genes important in tissue maintenance and disease. NGS brings sensitivity, specificity, and the ability to maximize data acquisition and minimize costs by using multiplex strategies to allow many samples to be sequenced simultaneously with small RNA analysis. This strategy has enabled researchers to perform genome- wide and high depth sequencing studies that would not be possible using other technologies. The ability to quickly and efficiently measure or profile these transcripts has great importance in research and clinical applications. 

However, small RNA sequencing has typically suffered from three major drawbacks: 1) severe bias, such that sequencing data does not reflect original miRNA abundances, 2) the need to gel purify final libraries, and 3) lack of low-input protocols. 

Bias is introduced into small RNA libraries during the ligation steps. Small RNA library preparation involves ligating adapters directly onto the 3’ and 5’ ends of the RNA molecule in two separate steps, and each of these ligation steps has been shown to introduce severe bias into library preparation. Recent studies have shown that inserting random bases at the ends of the adapters greatly reduces bias in comparison to using non-randomized adapters. 

Another major drawback of small RNA sequencing is the need to purify final libraries by gel, typically by PAGE gel. This need is due to the small difference in the size of adapter-dimer molecules versus insert-containing molecules following the PCR step of library preparation. In typical DNA or RNA library prep, insert-containing molecules are at least 100 bp larger than adapter-dimer molecules, and thus can be removed using SPRI magnetic beads. However, since insert-containing molecules are only ~20 bp larger than adapter-dimer molecules in small RNA libraries, SPRI size selection is not feasible, and gel-based selection must be performed. The need for gel-based size selection greatly limits both throughput and automation potential of small RNA library preparation, as only a limited number of libraries can be run on a single gel and it is a labor-intensive process that is not amenable to automation. 

The lack of low-input protocols for small RNA-Seq is also related to adapter-dimer formation. Small RNA sequencing is somewhat unique in that additional PCR cycles result in negligible bias; thus it should theoretically be possible to create low-input small RNA libraries by using a high number of PCR cycles. However, adapter-dimers present in the libraries will also be greatly amplified, which eventually leads to a library where adapter-dimer products are extremely abundant, making it difficult to isolate insert-containing products and leading to sequencing data where very few of the reads are useful. A number of methods have been developed to reduce adapter-dimer formation in small RNA library preparation, but unfortunately none are effective at reducing adapter-dimer formation to such an extent that gel-free or low-input small RNA library preparation is possible. 

In response to the drawbacks associated with small RNA sequencing, Bioo Scientific has developed a small RNA library preparation method that utilizes randomized adapters for greatly reduced bias and features gel-free or low-input protocols, which are possible due to significant reduction in adapter-dimer formation. This method is currently available as the NEXTflex™ Small RNA-Seq Kit v3.