Title | Generation of a Transcriptional Radiation Exposure Signature in Human Blood Using Long-Read Nanopore Sequencing. |
Publication Type | Journal Article |
Year of Publication | 2020 |
Authors | Cruz-Garcia, Lourdes, Grainne O'Brien, Botond Sipos, Simon Mayes, Michael I. Love, Daniel J. Turner, and Christophe Badie |
Journal | Radiat Res |
Volume | 193 |
Issue | 2 |
Pagination | 143-154 |
Date Published | 2020 Feb |
ISSN | 1938-5404 |
Keywords | Blood, Dose-Response Relationship, Radiation, Genomics, Humans, Leukocytes, Mononuclear, Nanopore Sequencing, Radiation Exposure, Transcription, Genetic, Transcriptome |
Abstract | In the event of a large-scale event leading to acute ionizing radiation exposure, high-throughput methods would be required to assess individual dose estimates for triage purposes. Blood-based gene expression is a broad source of biomarkers of radiation exposure which have great potential for providing rapid dose estimates for a large population. Time is a crucial component in radiological emergencies and the shipment of blood samples to relevant laboratories presents a concern. In this study, we performed nanopore sequencing analysis to determine if the technology can be used to detect radiation-inducible genes in human peripheral blood mononuclear cells (PBMCs). The technology offers not only long-read sequencing but also a portable device which can overcome issues involving sample shipment, and provide faster results. For this goal, blood from nine healthy volunteers was 2 Gy X irradiated. After PBMC isolation, irradiated samples were incubated along with the controls for 24 h at 37°C. RNA was extracted, poly(A)+ enriched and reverse-transcribed before sequencing. The data generated was analyzed using a Snakemake pipeline modified to handle paired samples. The sequencing analysis identified a radiation signature consisting of 46 differentially expressed genes (DEGs) which included 41 protein-coding genes, a long non-coding RNA and four pseudogenes, five of which have been identified as radiation-responsive transcripts for the first time. The genes in which transcriptional expression is most significantly modified after radiation exposure were APOBEC3H and FDXR, presenting a 25- and 28-fold change on average, respectively. These levels of transcriptional response were comparable to results we obtained by quantitative polymerase chain reaction (qPCR) analysis. exposure analyses showed a transcriptional radioresponse at 24 h postirradiation for both genes together with a strong dose-dependent response in blood irradiated . Finally, extrapolating from the data we obtained, the minimum sequencing time required to detect an irradiated sample using APOBEC3H transcripts would be less than 3 min for a total of 50,000 reads. Future improvements, in sample processing and bioinformatic pipeline for specific radiation-responsive transcript identification, will allow the provision of a portable, rapid, real-time biodosimetry platform based on this new sequencing technology. In summary, our data show that nanopore sequencing can identify radiation-responsive genes and can also be used for identification of new transcripts. |
DOI | 10.1667/RR15476.1 |
Alternate Journal | Radiat Res |
Original Publication | Generation of a transcriptional radiation exposure signature in human blood using long-read nanopore sequencing. |
PubMed ID | 31829904 |
PubMed Central ID | PMC7055010 |
Grant List | P01 CA142538 / CA / NCI NIH HHS / United States P30 ES010126 / ES / NIEHS NIH HHS / United States R01 HG009937 / HG / NHGRI NIH HHS / United States R01 MH118349 / MH / NIMH NIH HHS / United States |