Method for discriminating between live and dead microbes in a sample

A method for discriminating between live and dead microbes in a sample, comprising discriminating between transcriptionally-active and inert microbial nucleic acid sequences in the sample, wherein the method comprises the steps of:

• (a) sequencing a set of RNAs extracted from the sample, wherein the set of RNAs is obtained by culturing the sample in the presence of an RNA-labelling agent and further by submitting the extracted RNAs to conditions promoting thymidine (T)-to-cytidine (C) substitutions;
• thereby obtaining a set of sequence reads;
• (b) determining a consensus microbial nucleic acid sequence through:
• (i) aligning the set of sequence reads or contigs onto a database comprising microbial nucleic acid sequences, and
• (ii) deriving a consensus microbial nucleic acid sequence assembled de novo from said sequence reads that have mapped with an expected value (e-value) below 10−2 to a microbial nucleic acid sequence of the database;
• (c) determining the number and/or rate of substituted nucleotides in the set of sequence reads that have mapped with an e-value below 10−2 to a microbial nucleic acid sequence of the database at step (b) in comparison with the consensus microbial nucleic acid sequence assembled de novo at step (b);
• and
• (d) concluding that the consensus microbial nucleic acid sequence belongs to a live microbe if, of the set of sequence reads that have mapped with an e-value below 10−2 to a microbial nucleic acid sequence of the database at step (b), the number and/or rate of T-to-C substitutions determined in step (c) is greater than the average number and/or rate of substitutions of all other nucleotides in the same sequence reads,
• wherein steps (c) and (d) do not require the use of an external microbial nucleic acid sequence reference.