IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution

Besides canonical microRNAs (miRNAs), sequence-based variations called isomiRs have biological relevance and diagnostic potential; however, accurate calling of these post-transcriptional modifications is challenging, especially for low input samples. Here, we present IsoSeek, a sequencing protocol that reduces ligation and PCR amplification bias and improves the accuracy of miRNA detection in low input samples. We describe steps for using randomized adapters combined with unique molecular identifiers (UMI), library quantification, and sequencing, followed by detailed procedures for data processing and analysis. For complete details on the use and execution of this protocol, please refer to C. Gomez-Marti ' n et al. (2023)1 and Van Eijndhoven et al. (2021).2

Automated summary

This article presents IsoSeek, a sequencing protocol that improves the accuracy of miRNA detection in low input samples by reducing ligation and PCR amplification bias. The article provides detailed steps for using randomized adapters combined with unique molecular identifiers (UMI), library quantification, sequencing, and data processing and analysis.