Metagenomics

Metagenomics

Virology

Metagenomics is a broad-range test that utilises deep sequencing to sequence total RNA and total DNA in a clinical specimen; this has the potential for un-targeted pan-pathogen detection. Metagenomics 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. In CSF, the sensitivity is similar to targeted real-time PCR for both RNA and DNA pathogens. In tissue, the sensitivity for RNA viruses is similar to targeted real-time PCR. For DNA viruses, bacteria and fungi the sensitivity in tissue is reduced (approximately 100-fold) compared to targeted real-time PCR but comparable to pan-bacterial PCR (16S rRNA gene). Low level DNA pathogens in tissue may not be detected

Metagenomics is currently offered for tissue biopsies and CSF. The recommended specimen type is fresh tissue collected directly into RNALater. See below for collection and storage requirements:

1. CSF 1 ml (minimum 500 μl) whole CSF (not filtered or centrifuged). We recommend CSFs are stored at −80 °C within 24 hours of collection (maximum 72 hours) to minimise RNA degradation. CSF is not stable at room temperature. CSF collected with RNALater is not recommended due to poor sequence outcomes associated with dilution of the CSF. Smaller volumes, sub-optimal storage or any pre-processing (e.g. filtering or centrifugation) will reduce ability to detect pathogen

2. Fresh tissue - RNALater Specimens must be collected directly into RNALater at the point of collection (e.g. in theatre); this is to rapidly stabilise RNA and prevent degradation. Specimens stabilised in RNALater can be stored at +4 °C for up to one month prior to shipping and are stable at room temperature for up to 1 week. Ready to use pre-aliquoted RNALater can be purchased directly from Thermo Fisher Scientific (catalogue number AM7022). Aliquots of RNALater are also available on request from GOSH Microbiology/Virology labs. Please note only RNALater has been validated for or processing methods therefore we do not advise the use of other RNA stabilisation solution

3. Fresh tissue - Frozen Specimens that are not stabilised in RNALater should be immediately collected onto dry ice (e.g. in theatre) 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 immediately stabilised in RNALater nor frozen is not recommended for metagenomics due to rapid degradation of RNA. Tissue in saline is not suitable. Please be aware sub-optimal specimen collection, storage or transport will result in RNA degradation which will significantly reduce ability to detect RNA pathogens. FFPE (formalin fixed paraffin embedded) tissue 8x 10 μm or 4 x 20 μm rolled sections. Please be aware the ability to detect low-level pathogens will be reduced in FFPE tissue compared to fresh tissue due to degradation of nucleic acid during formalin fixation. FFPE is a sub-optimal specimen type but can be processed in the absence of fresh tissue

Specimen Transport conditions:

1. Tissue in RNALater: Ambient temperature

2. FFPE: Ambient temperature

3. Frozen tissue: Dry ice

4. CSF: Dry ice

2 weeks

Not applicable

No