RNASeq pathogen detection – Service Summary


RNASeq is a technique that utilises metagenomics (deep sequencing) to sequence total RNA in a clinical specimen; this has the potential for un-biased pan-pathogen detection.

RNASeq is particularly useful in difficult-to-diagnose syndromes, such as encephalitis, in which an infectious aetiology is suspected but cannot be identified by routine methods.

This technique has been optimised for detection of RNA viruses. DNA viruses are detectable via RNA transcripts, however there is a potential limited ability to detect DNA pathogens if the level of replication is low. Detection of bacteria and fungi is possible, but not clinically validated therefore for pan-bacterial and pan-fungal detection please see our Molecular Bacteriology service (http://www.labs.gosh.nhs.uk/media/760967/RequestFormPCR.pdf).

Please note this test is under on-going validation and as such is not yet accredited by UKAS against ISO 15189:2012.

 

Test Description

Specimen required

NHS Price

Turnaround Time

Request Form

RNASeq

Tissue biopsy (fresh/frozen or FFPE)

£1350

2-4 weeks

A

 

Specimen Requirements

 

RNASeq is currently only offered for tissue biopsies. See below for tissue collection and storage requirements.

 

Fresh tissue  -RNALater

We recommend specimens are collected directly into RNALater at the point of collection; this is to rapidly stabilise RNA and prevent degradation. Specimens stabilised in RNALater can be stored at room temperature for up to 1 week or at +4 °C for up to one month prior to shipping.

Aliquots of RNALater are available on request or from laboratory reagent suppliers.

 

Fresh tissue  -Frozen

Specimens that are not stabilised in RNALater should be immediately collected onto dry ice and stored at −80 °C prior to shipping. It is important to avoid any freeze-thaw cycles.

 

Fresh tissue - unstabilised

Tissue that has not been stabilised in RNALater nor frozen is not recommended for RNASeq due to degradation of RNA.

Please be aware sub-optimal specimen collection, storage or transport will result in RNA degradation which may reduce ability to detect pathogens.

 

FFPE (formalin fixed paraffin embedded) tissue

4x 10 µm or 2 x 20 µm rolled sections

Please be aware the ability to detect low-level pathogens may be reduced in FFPE tissue compared to fresh tissue due to degradation of nucleic acid during formalin fixation. 

 

Specimen transport

 

Tissue in RNALater: Ambient temperature or cold (refrigerated/ice pack)

Frozen tissue: Dry ice

 FFPE: Ambient temperature

 

Coming Soon

  • DNA sequencing - for improved detection of DNA pathogens
  • Sequencing of CSF and other low-biomass specimens

 

Please send samples accompanied by request form to:

Virology Laboratory

Level 4, Camelia Botnar Laboratories 

Great Ormond Street Hospital for Children
Great Ormond Street
London WC1N 3JH

Hays Dx: GOSH DX 6640203, Bloomsbury 91 WC


General and Technical Enquiries:  

Dr Julianne Brown

Tel: 020 7405 9200 Ext 5929

Email: Julianne.Brown@nhs.net


Clinical Enquiries:

Professor Judy Breuer

Tel: 020 7405 9200 Ext 42129 / 020 3108 2130
Email: j.breuer@ucl.ac.uk

 

Relevant publications arising from RNASeq service in our laboratory

  1. Julianne R Brown, Tehmina Bharucha and Judith breuer (2018). Encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases, Journal of Infection, In press, DOI: 10.1016/j.jinf.2017.12.014
  2. Sofia Morfopoulou, Edward T. Mee, Sarah M. Connaughton, Julianne R. Brown, Kimberly Gilmour, WK 'Kling' Chong, W. Paul Duprex, Deborah Ferguson, Mike Hubank, Ciaran Hutchinson, Marios Kaliakatsos, Stephen McQuaid, Simon Paine, Vincent Plagnol, Christopher Ruis, Alex Virasami, Hong Zhan, Thomas S. Jacques, Silke Schepelmann, Waseem Qasim, Judith Breuer (2016). Deep sequencing reveals persistence of cell-associated mumps vaccine virus in chronic encephalitis, Acta Neuropathologica, 133(1): 139-147.
  3. Lum SH, Turner A, Guiver M, Bonney D, Martland T, Davies E, Newbould M, Brown J, Morfopoulou S, Breuer J, Wynn R (2016). An emerging opportunistic infection: fatal astrovirus (VA1/HMO-C) encephalitis in a pediatric stem cell transplant recipient, Transplant Infectious Disease, 18(6): 960-964
  4. Morfopoulou S, Brown JR, Davies EG, Anderson G, Virasami A, Qasim W, Chong WK, Hubank M, Plagnol V, Desforges M, Jacques TS, Talbot PJ, Breuer J (2016). Coronavirus HCoV-OC43 Associated with Fatal Encephalitis, New England Journal of Medicine, 375(5): 497-8.
  5. Duncan JAD, Mohamad SMB, Young DF, Skelton AJ, Leahy TR, Munday DC, Butler KM, Morfopoulou S, Brown JR, Hubank M, Connell J, Gavin PJ, McMahon C, Dempsey E, Lynch NE, Jacques TS, Valappil M, Cant AJ, Beuer J, Engelhardt KR, Randall RE and Hambleton S (2015). Human IFNAR2 deficiency: Lessons for antiviral immunity, Science Translational Methods, 7 (307).
  6. Brown JR, Morfopoulou S, Hubb J, Emmett WA, Ip W, Shah D, Brooks T, Paine SML, Anderson G, Virasami A, Tong CY W, Clark DA, Plagnol V, Jacques TS, Qasim W, Hubank M, Breuer J (2015). Astrovirus VA1/HMO-C: an increasingly recognised neurotropic pathogen in immunocompromised patients. Clinical Infectious Diseases, 60 (6): 881-888

 

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