Generation of a Transcriptional Radiation Exposure Signature in Human Blood Using Long-Read Nanopore Sequencing

Cruz-García, Lourdes; O’Brien, Gráinne; Sipos, Botond; Mayes, Simon; Love, Michael I.; Turner, Daniel J.; Badie, Christophe

doi:10.1667/rr15476.1

Cited by 30 publications

(24 citation statements)

References 61 publications

(81 reference statements)

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“…Amongst them, seven variants presented a significant response to IR, FDXR-201 as expected and FDXR-204, FDXR-206, FDXR-208, FDXR-213, FDXR-218, and FDXR-219 ( Figure 6 ). Amongst the seven, FDXR-206, FDXR-218, and FDXR-219 were very low expressed while FDXR-218 and FDXR-219 remarkably showed an undetectable endogenous levels (no counts presented in the control sample), even after generating over 50 million reads of mRNA per sample [ 28 ] ( Figure 6 ).…”

Section: Resultsmentioning

confidence: 99%

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In Vivo Validation of Alternative FDXR Transcripts in Human Blood in Response to Ionizing Radiation

Cruz-García

O’Brien

Sipos

et al. 2020

IJMS

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Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected; moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers.

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Section: Resultsmentioning

confidence: 99%

“…After the cDNA synthesis, a selective PCR amplification was performed for full-length transcripts before adding specific adapters to start the sequencing run in the PromethION sequencer. Details can be found in Cruz-Garcia et al (2020) [28].…”

Section: Nanopore Sequencing Analysismentioning

confidence: 99%

In Vivo Validation of Alternative FDXR Transcripts in Human Blood in Response to Ionizing Radiation

Cruz-García

O’Brien

Sipos

et al. 2020

IJMS

Self Cite

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“…transplantation status) (Harbron et al, 2018a), together with improved dose assessment (Harbron et al, 2016) and overall risk of cancers (Journy et al, 2016(Journy et al, , 2017Harbron et al, 2017aHarbron et al, , 2018a for low-dose medically exposed populations. In the area of radiobiology, key recent outputs include development and validation of cytogenetic and genetic biomarkers of radiation exposure in medically exposed populations to underpin dose assessment (Cruz-Garcia et al, 2018;Einbeck et al, 2018;Moquet et al, 2018;O'Brien et al, 2018;Tichy et al, 2018), development of a new method of premature chromosome condensation to increase the speed of biological assessment of higher doses (Sun et al, 2019(Sun et al, , 2020, development of a new protocol for rapid gene-expression-based dose estimation (Polozov et al, 2019), and identification of further new genes suitable for biodosimetric purposes using rapid long-read DNA sequencing methods (Cruz-Garcia et al, 2020). Other major contributions include publication of peer-reviewed papers focused on the dose to the lens of the eye following CT scan exposures (Harbron et al, 2019), and the limited impact of iodinated contrast media on doses to haematopoietic stem cells (Harbron et al, 2017b(Harbron et al, , 2018b.…”

Section: Nihr Hpru Radiation Themementioning

confidence: 99%

The 2019 Bo Lindell Laureate Lecture: On the use of interdisciplinary, stakeholder-driven, radiation protection research in support of medical uses of ionising radiation

Ainsbury

2020

Ann ICRP

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Medical exposures form the largest manmade contributor to total ionising radiation exposure of the UK population. In recent years, new technologies have been developed to improve treatment and prognosis of individuals treated with radiation for diseases such as cancer. However, there is evidence of public, patient, and medical professional concern that radiation protection regulations and practices, as well as understanding of potential long-term adverse health effects of radiation exposure (in the context of other health risks), have not always ‘kept pace’ with technological developments in this field. This is a truly complex, multi-disciplinary problem for the modern world. The ‘Radiation Theme’ of the Public Health England and Newcastle University Health Protection Research Unit on ‘Chemical and Radiation Threats and Hazards’ is addressing this need, with a key focus on a genuinely interdisciplinary approach bringing together world-leading epidemiologists, radiation biologists, clinicians, statisticians, and artists. In addition, the project has a strong grounding in public, patient, and medical professional involvement in research. Similarly, the EU-CONCERT-funded LDLensRad project seeks to understand the mechanisms of action of low-dose ionising radiation in the lens of the eye, and the potential contribution to the development of cataract – in contemporary research, such projects will only be considered successful when they make use of expertise from a variety of fields and when they are able to demonstrate that the outputs are not only of benefit to society, but that society understands and welcomes the benefits. Finally, successful engagement, training, and retention of early career scientists within this field is crucial for sustainability of the research. Herein, the contribution of embedded interdisciplinary working, stakeholder involvement, and training of early career scientists to recent advancements in the field of medical (and wider) radiation protection research is discussed and considered.

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“…Cruz-Garcia et al. ( 17 ) used a Snakemake pipeline which included the edgeR ( 18 ) quasi-likelihood method to identify a radiation exposure signature consisting of 46 transcripts from a high coverage (40–75 million reads per sample) ONT PromethION PCR cDNA dataset of human blood cells.…”

Section: Introductionmentioning

confidence: 99%

The long and the short of it: unlocking nanopore long-read RNA sequencing data with short-read differential expression analysis tools

Dong

Tian

Gouil

et al. 2021

NAR Genomics and Bioinformatics

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Application of Oxford Nanopore Technologies’ long-read sequencing platform to transcriptomic analysis is increasing in popularity. However, such analysis can be challenging due to the high sequence error and small library sizes, which decreases quantification accuracy and reduces power for statistical testing. Here, we report the analysis of two nanopore RNA-seq datasets with the goal of obtaining gene- and isoform-level differential expression information. A dataset of synthetic, spliced, spike-in RNAs (‘sequins’) as well as a mouse neural stem cell dataset from samples with a null mutation of the epigenetic regulator Smchd1 was analysed using a mix of long-read specific tools for preprocessing together with established short-read RNA-seq methods for downstream analysis. We used limma-voom to perform differential gene expression analysis, and the novel FLAMES pipeline to perform isoform identification and quantification, followed by DRIMSeq and limma-diffSplice (with stageR) to perform differential transcript usage analysis. We compared results from the sequins dataset to the ground truth, and results of the mouse dataset to a previous short-read study on equivalent samples. Overall, our work shows that transcriptomic analysis of long-read nanopore data using long-read specific preprocessing methods together with short-read differential expression methods and software that are already in wide use can yield meaningful results.

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Generation of a Transcriptional Radiation Exposure Signature in Human Blood Using Long-Read Nanopore Sequencing

Abstract: BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses.

Cited by 30 publications

References 61 publications

In Vivo Validation of Alternative FDXR Transcripts in Human Blood in Response to Ionizing Radiation

In Vivo Validation of Alternative FDXR Transcripts in Human Blood in Response to Ionizing Radiation

The 2019 Bo Lindell Laureate Lecture: On the use of interdisciplinary, stakeholder-driven, radiation protection research in support of medical uses of ionising radiation

The long and the short of it: unlocking nanopore long-read RNA sequencing data with short-read differential expression analysis tools

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