Biology subjects

Spengler, R. M.

Publications and source records attributed to Spengler, R. M..

2 recordsLinked to original sources

bioRxiv2019

Phospho-sRNA-seq reveals extracellular mRNA/lncRNA fragments as potential biomarkers in human plasma

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Whereas extracellular microRNAs (miRNAs) in blood plasma are extensively characterized, extracellular messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are less well-studied. We report that plasma contains fragmented mRNAs and lncRNAs that are largely missed by standard small RNA-seq protocols due to lack of 5 phosphate or presence of 3 phosphate. These fragments were revealed using a modified protocol ("phospho-sRNA-seq") incorporating RNA treatment with T4-polynucleotide kinase, which we compared with standard small RNA-seq for sequencing synthetic RNAs with varied 5 and 3 ends, as well as human plasma exRNA. Analyzing phospho-sRNA-seq data using a custom, high-stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue-specific gene sets. In a longitudinal study of bone marrow transplant patients, bone marrow-and liver-enriched exRNA genes tracked with bone marrow recovery and liver injury, respectively, providing proof-of-concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho-sRNA-seq opens up new possibilities for plasma transcriptomic biomarker development.

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↗