Some information on the protocol we used at the Geneva University Hospitals.
We used an unbiased mNGS approach directly on the clinical sample (plasma) as follows:
Virus nucleic acid extractions done on a eMAG instrument (bioMérieux) (initial sample volume = 400 µl, elution =50 µl). 10 µl of eluate (the same as that used to perform the rRT-PCR analysis) was used to construct the library. Ribosomal RNA was removed using the Ribo-Zero Gold depletion kit (Illumina) prior to the preparation of the library (TruSeq total RNA preparation protocol, Illumina). Library was run on a MiSeq instrument (2 × 100-bp protocol).
Bioinformatic Analysis :
- Trimming/Filtering: trimmomatic Version=0.36
- Dehosting: snap-aligner Version=1.0beta.18, DB:=gencode_v43
- Assembly: IDBA-UD Version=1.1.3
- Remaping : snap-aligner Version=1.0beta.18
Total reads = 24936538 (56.608 % Human RNA)
ANDV/Switzerland/Hu-3337/2026 sequence:
Segment reads median depth
L 67979 766
M 48785 954
S 17976 739