Figure 2. Quantitative validation of AQRNA-seq.

(a) Oligonucleotide spike-ins demonstrate a linear relationship between copy number and read count. Oligonucleotides 25–80 nt long were subjected to AQRNA-seq at different concentrations (GEO accession GSE139936). Dot plots show all data, bar denoting mean, for N=3 experiments. (b) Minimal sequence bias in AQRNA-seq analysis of the 963 miRNA Miltenyi miRXplore Universal Reference (GEO accession GSE139936). Among measured miRNAs, the 5’ and 3’ nucleotides were tabulated and their proportions plotted. Dot plot shows data for N=3 experiments on Day 1 and N=1 experiment on Day 2, with a dash denoting expected proportions of A, C, G, and U at each end among all 963 reference miRNAs. (c) AQRNA-seq quantitative fidelity was assessed using the miRXplore Reference (GEO accession GSE139936). Sequencing reads for each miRNA were normalized to expected values and sorted into 5 bins as denoted in the graph. The colored bar indicates the percentage of reads within 2- and 10-fold of expected abundance. (d) Comparison of the quantitative accuracy of miRNA libraries prepared from the miRXplore Reference using the AQRNA-seq (“AQ”) protocol and the following small RNA or miRNA library kits: Illumina TruSeq (ILM), Lexogen (LEX), NEBNext for Illumina (NEB), Perkin Elmer NextFlex (PEB), QIAseq miRNA (QIA), and Trilink CleanTag (TRI). Data for the kits was derived from Herbert et al.²⁶ For each replicate and for each kit, the percentage of total miRNAs found in each bin denoted in panel c was calculated. Dot plot shows data for N=3 experiments (ILM, LEX, TRI, AQ) or N=4 (NEB, PEB, QIA), with bars denoting mean ± SD. (e) Among AQRNA-seq and the other RNA-seq kits, a positive correlation exists between the average number of sequence variants detected and the percentage of miRNAs quantified within 2-fold of expected value. Sequence variants are defined as additions and subtractions to the insert sequences during library preparation; see Supplementary Figure 2 for the set of sequence variants arising for the kits. Data represent mean ± SD for N=3 experiments (ILM, LEX, TRI, AQ) or N=4 (NEB, PEB, QIA). (f) Correlation of tRNA quantification results using AQRNA-seq versus data derived from 2D gel electrophoresis and northern blotting by Dong et al.²⁷ Data represent individual values derived from Dong et al.²⁷ plotted relative to mean values from N=3 AQRNA-seq analyses of E. coli tRNAs.