Abstract
Highly abundant microRNAs (miRNAs) in small RNA sequencing libraries make it difficult to obtain efficient measurements of more lowly expressed species. We present a new method that allows for the selective blocking of specific, abundant miRNAs during preparation of sequencing libraries. This technique is specific with little off-target effects and has no impact on the reproducibility of the measurement of non-targeted species. In human plasma samples, we demonstrate that blocking of highly abundant hsa-miR-16–5p leads to improved detection of lowly expressed miRNAs and more precise measurement of differential expression overall. Furthermore, we establish the ability to target a second abundant miRNA and to multiplex the blocking of two miRNAs simultaneously. For small RNA sequencing, this technique could fill a similar role as do ribosomal or globin removal technologies in messenger RNA sequencing.
Highlights
The measurement of small RNAs, and in particular microRNAs, is an important tool in many biological fields due to their key roles in fundamental cellular processes such as cell fate maintenance, DNA damage response, cell-cycle regulation and others. miRNAs primarily function as modulators of specific mRNA levels by targeting various protein complexes (RISC and others) to mRNA through complementary base pairing
The measurement of small RNAs, and in particular microRNAs, is an important tool in many biological fields due to their key roles in fundamental cellular processes such as cell fate maintenance, DNA damage response, cell-cycle regulation and others (reviewed in [1,2,3]). miRNAs primarily function as modulators of specific mRNA levels by targeting various protein complexes (RISC and others) to mRNA through complementary base pairing (reviewed in [4])
In a set of small RNA sequencing libraries from 27 human plasma samples that we prepared by using a slightly modified version of the protocol described by Alon et al [28], we observed that reads mapping to hsa-miR-16–5p comprised between and 60% of the total aligned reads in the Nucleic Acids Research, 2015, Vol 43, No e145
Summary
The measurement of small RNAs, and in particular microRNAs (miRNAs), is an important tool in many biological fields due to their key roles in fundamental cellular processes such as cell fate maintenance, DNA damage response, cell-cycle regulation and others (reviewed in [1,2,3]). miRNAs primarily function as modulators of specific mRNA levels by targeting various protein complexes (RISC and others) to mRNA through complementary base pairing (reviewed in [4]). Next-generation DNA sequencing (NGS) is a powerful method for the discovery and quantification of small RNAs due to its technical performance [26], low expense, ultra-high throughput and its ability to agnostically detect and measure new species. NGS of small RNAs has several technical challenges Among these is the well-reported biased behavior of the modified forms of T4 RNA Ligase 2 commonly used in sequencing library generation protocols. This bias manifests in small RNA libraries as differential ligation, creating an over-representation of certain species and an underrepresentation of others [27,28,29,30,31]. Highly abundant species interfere with many normalization techniques, limiting the utility of the collected reads
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