Etheridge, A. | bioRxiv Science

bioRxiv2017

Isolation of nucleic acids from low biomass samples: detection and removal of sRNA contaminants

Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. Due to its inherent instability, contamination with RNA is usually considered to be unlikely. Here we report the presence of small RNA (sRNA) contaminants in widely used microRNA extraction kits and means for their depletion. Sequencing of sRNAs extracted from human plasma samples was performed and significant levels of non-human (exogenous) sequences were detected. The source of the most abundant of these sequences could be traced to the microRNA extraction columns by qPCR-based analysis of laboratory reagents. The presence of artefactual sequences originating from the confirmed contaminants were furthermore replicated in a range of published datasets. To avoid artefacts in future experiments, several protocols for the removal of the contaminants were elaborated, minimal amounts of starting material for artefact-free analyses were defined, and the reduction of contaminant levels for identification of bona fide sequences using ultra-clean extraction kits was confirmed. In conclusion, this is the first report of the presence of RNA molecules as contaminants in laboratory reagents. The described protocols should be applied in the future to avoid confounding sRNA studies.

molecular biology↗

bioRxiv2017

Accuracy, Reproducibility And Bias Of Next Generation Sequencing For Quantitative Small RNA Profiling: A Multiple Protocol Study Across Multiple Laboratories

Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.

genomics↗