Computational protocol: Serum microRNA signatures as "liquid biopsies" for interrogating hepatotoxic mechanisms and liver pathogenesis in human

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Protocol publication

[…] RNA samples were prepared for sequencing using the TruSeq Small RNA Preparation Kit (Illumina) and subsequently sequenced using the Illumina HiSeq 2000 sequencing platform (GEO accession GSE90028 and GSE59565). After quality control using FastQC (version 0.11.3) the data were processed using miRDeep2 (v2.0.0.5) [] and miRBase (release 21) [] as described earlier []. [...] The quantitative miRNA levels retrieved from the miRDeep2 output were analyzed using R (version 3.2.2). To detect differential miRNA levels among the conditions we used the package DESeq2 (version 1.8.1) []. Therefore we tested each condition (for the APAP cases only the earliest time-points after overdosing were considered) separately against the HC. The results were filtered for significantly altered miRNAs applying a false discovery rate below 5 percent as described previously []. For isomiR quantification we used Isomirage [] to map all reads to an isomiR database derived from miRBase. The reads were converted to proportions and for each condition the predominant isomiR per miRNA was identified. Based on the remaining sequencing reads miRDeep2 was used to predict novel miRNAs. Briefly, after filtering for other RNA species, the remaining reads were mapped onto the human genome. Subsequently, the results were checked for a valid ratio between precursor and mature read counts and for the possibility of forming a hairpin structure. […]

Pipeline specifications

Software tools FastQC, miRDeep, DESeq2, IsomiRage
Databases miRBase
Application sRNA-seq analysis
Diseases Diabetes Mellitus, Hepatitis B, Liver Diseases, Machado-Joseph Disease, Chemical and Drug Induced Liver Injury, Drug Overdose, Drug-Related Side Effects and Adverse Reactions
Chemicals Acetaminophen, Alanine