Quantitative mapping of the cellular small RNA landscape with AQRNA-seq

Abstract

Current next-generation RNA sequencing methods do not provide accurate quantification of small RNAs within a sample due to sequence-dependent biases in capture, ligation, and amplification during library preparation. We present a method, Absolute Quantification (AQ) RNA-seq, that minimizes biases and provides a direct, linear correlation between sequencing read count and copy number for all small RNAs in a sample. Library preparation and data processing were optimized and validated using a 963-member miRNA reference library, oligonucleotide standards of varying lengths, and northern blots. Application of AQRNA-seq to a panel of human cancer cells revealed >800 detectable miRNAs that varied during cancer progression, while application to bacterial tRNA pools, with the challenges of secondary structure and abundant modifications, revealed 80-fold variation in tRNA isoacceptor levels, stress-induced site-specific tRNA fragmentation, quantitative modification maps, and evidence for stress-induced tRNA-driven codon-biased translation. AQRNA-seq thus provides a versatile means to quantitatively map the small RNA landscape in cells.

5′ Half of specific tRNAs feeds back to promote corresponding tRNA gene transcription in vertebrate embryos

“…S3, A and B). Similar to tRNA expression in other systems (43)(44)(45), however, some tRNA genes showed severely biased expression levels in zebrafish embryos. For example, 75 to 86% of total tRNAs at any examined developmental stages were tRNA His/GTG that has 511 gene copies in the genome (Fig.…”

Aberration-corrected ultrafine analysis of miRNA reads at single-base resolution: a k-mer lattice approach

“…Our algorithm was tested on the sequencing reads of an equimolar mixture of synthetic miRNAs from the miRXplore Universal Reference that consists of 963 miRNAs from human, mouse, rat and viral sources (three replicate samples miRXploreUR rep1-3 corresponding to GSE139936.GSM4149813, GSE139936.GSM4149814 and GSE139936.GSM4149815 ( 51 )). The test was to verify…”

The Aminoacyl-tRNA Synthetase and tRNA Expression Levels Are Deregulated in Cancer and Correlate Independently with Patient Survival

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