Detection of guest reads in Dual RNA-Seq

Question

[yoshifumimiya]

I attempted a "Dual RNA-Seq" on a nasal sample from a person with RNA virus infection. The RNA virus infection was confirmed by PCR.

However, RNA-Seq sometimes detects guest reads and sometimes does not.

According to the reference [1], the sensitivity of Dual RNA-Seq seems to be 86%.

The reason why it cannot be detected is that

(1) Appearance of unanalyzable specimens due to the effect of new mutations

→ Is it possible to change the option to allow for mutations when mapping?

(2) Decreased detection sensitivity in samples with low viral nucleic acid content.

→ Is PCR more sensitive for detection?

Are there any other reasons why guest reads cannot be detected?

Your comments would be appreciated. Thank you in advance.

Reference [1]: Burgess DJ. Gene expression: host-pathogen duels revealed by dual RNA-seq in vivo. Nat Rev Genet. 2017 Feb 15;18(3):143. doi 10.1038/nrg.2017.10. PMID: 28196982.

Answer 1

[Trivas]

PCR is more sensitive than sequencing. What kind of selection are you doing for your RNA libraries - are you sure your RNA virus is polyadenylated if you're going that route? Is there weird secondary structure that could inhibit 1st strand synthesis? You could try a metagenomic/assembly type approach with your unmapped host reads to see if you can assemble your viral genome, or you could allow more mismatches in your mapping step, but I would suggest thinking about whether the chemistry of your library prep makes sense for your system as a starting point.

[yoshifumimiya]

Thank you for your prompt reply.

I was using a public database, so I checked the METHOD and found that "The swab was transferred to a tube containing media supplemented with 13 μM dithiothreitol to inactivate the virus."

You are correct, the chemistry of library prep was not good.

Thank you for your valuable input. It was very helpful.