High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency

Abstract

The systemic evolution of ligands by exponential enrichment (SELEX) technique is a powerful and effective aptamer-selection procedure. However, modifications to the process can dramatically improve selection efficiency and aptamer performance. For example, droplet digital PCR (ddPCR) has been recently incorporated into SELEX selection protocols to putatively reduce the propagation of byproducts and avoid selection bias that result from differences in PCR efficiency of sequences within the random library. However, a detailed, parallel comparison of the efficacy of conventional solution PCR versus the ddPCR modification in the RNA aptamer-selection process is needed to understand effects on overall SELEX performance. In the present study, we took advantage of powerful high throughput sequencing technology and bioinformatics analysis coupled with SELEX (HT-SELEX) to thoroughly investigate the effects of initial library and PCR methods in the RNA aptamer identification. Our analysis revealed that distinct “biased sequences” and nucleotide composition existed in the initial, unselected libraries purchased from two different manufacturers and that the fate of the “biased sequences” was target-dependent during selection. Our comparison of solution PCR- and ddPCR-driven HT-SELEX demonstrated that PCR method affected not only the nucleotide composition of the enriched sequences, but also the overall SELEX efficiency and aptamer efficacy.

AptaTRACE Elucidates RNA Sequence-Structure Motifs from Selection Trends in HT-SELEX Experiments

“…Next, we use simulated data, produced with an extended version of our AptaSim program (Hoinka et al, 2015) developed for this study, to compare the performance of AptaTRACE to other methods that can handle similar data sizes or incorporate secondary structure into their models. Finally, we show our results of applying AptaTRACE to an in vitro selection consisting of high-throughput data from 9 rounds of cell-SELEX (Takahashi et al, 2016). …”

Applications of High-Throughput Sequencing for In Vitro Selection and Characterization of Aptamers

“…Furthermore, a clustering between k-mers enriched in the same predicted structure context is performed to identify consensus motifs. To validate AptaTrace, Dao et al used data from a cell-SELEX previously performed to identify aptamers against the C-C chemokine receptor type 7 (CCR7) [37]. A CUGUG motif highly represented in an apical loop structure was found and two mutants of this family (C1-A and C2-A) demonstrated binding by flow cytometry assays.…”

“…The effect of PCR on the SELEX process has also been study using HTS by Takahashi et al [37]. Two cell-SELEX were performed against CCR7.…”

Ideal‐filter capillary electrophoresis: A highly efficient partitioning method for selection of protein binders from oligonucleotide libraries

“…The multiround selection is resource‐consuming and time‐consuming. In multiround selection of oligonucleotide aptamers via the systematic evolution of ligands by exponential enrichment (SELEX) process (repetition of binding/partitioning and PCR amplification), the number of rounds is theoretically unlimited, but a large number of rounds can lead to selection failure due to sequence biases of polymerases . In selection of binders from DELs, the number of rounds is limited to three or four due to (i) binder loss in partitioning and (ii) the inability to PCR‐amplify the binders, which are not oligonucleotides in this case (e.g., small molecules) .…”