Biology subjects

Barczak, A. J.

Publications and source records attributed to Barczak, A. J..

3 recordsLinked to original sources

bioRxiv2019

Comparison of miRNA profiling methods using synthetic miRNA pools and standardized exRNA samples reveals substantial performance differences

MicroRNAs (miRNAs) found in biofluids play functional roles in health and in disease pathogenesis, underpinning their potential as clinical biomarkers. Several platforms for measurement of extracellular RNAs have recently become available. We evaluated the reproducibility, accuracy, sensitivity, and specificity of four miRNA quantification platforms, including one widely used discovery approach (small RNA-seq) and three targeted platforms (FirePlex, EdgeSeq, and nCounter). Using pools of synthetic miRNAs, we observed that reproducibility was highest for RNA-seq and EdgeSeq, that all three targeted platforms had lower bias than RNA-seq, and that RNA-seq had the best ability to distinguish between present and absent sequences. Overall reproducibility was lower for plasma samples than synthetic miRNA pools. We compared expression of placental miRNAs in plasma from pregnant and non-pregnant women and observed expected differences with RNA-seq and EdgeSeq, but not FirePlex or nCounter. We conclude that differences in performance among miRNA profiling platforms impact their relative utility as potential assay systems for clinical biomarkers.

genomics↗

bioRxiv2018

Large differences in small RNA composition between human biofluids

Extracellular miRNAs and other small RNAs are implicated in cellular communication and may be useful as disease biomarkers. We systematically compared small RNAs in 12 human biofluid types using RNA-seq. miRNAs and tRNA-derived RNAs (tDRs) accounted for the majority of mapped reads in all biofluids, but the ratio of miRNA to tDR reads varied from 72 in plasma to 0.004 in bile. miRNA levels were highly correlated across all biofluids but levels of some miRNAs differed markedly between biofluids. tDR populations differed extensively between biofluids. Y RNA fragments were seen in all biofluids and accounted for >10% of reads in blood plasma, serum, and CSF. Reads mapping exclusively to piRNAs were very rare except in seminal plasma. These results demonstrate extensive differences in small RNAs between human biofluids and provide a useful resource for investigating extracellular RNA biology and developing biomarkers.

genomics↗

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↗