Comparison of mNGS results to expected standard-of-care results

SampleExpected standard-of-care resultmNGS resultsa
PelletNeat CSFSupernatant
Subsample I (no BB, cDNA)Subsample J (BB, DNAamp)bSubsample L (no BB, cDNA)Subsample M (BB, DNAamp)Subsample F (no BB, cDNA)Subsample G (BB, DNAamp)c
1Varicella-zoster virusHuman herpesvirus 3 (GC, 0.3%; UKM, 602), Raoultella ornithinolytica, Corynebacterium singulareHuman herpesvirus 3 (GC, 32.7%; UKM, 33,001)Human herpesvirus 3 (GC, 1.9%; UKM, 2,319)Human herpesvirus 3 (GC, 43.7%; UKM, 45,575)Human herpesvirus 3 (GC, 4.0%; UKM, 4,503), Polaromonas naphthalenivoransHuman herpesvirus 3 (GC, 33.9%; UKM, 37,640), Cronobacter dublinensis
2Cryptococcus neoformansCryptococcus neoformans (GC, 10.4%; UKM, 209,888), TTVCryptococcus neoformans (GC, 70.1%; UKM, 1,229,464), Cryptococcus gattiiCryptococcus neoformans (GC, 3.2%; UKM, 66,648), TTVCryptococcus neoformans (GC, 62.3%; UKM, 1,125,775), Cryptococcus gattiiTTVCryptococcus neoformans (GC, 73.5%; UKM, 136,619), Cryptococcus gattii
3Pseudomonas aeruginosaPositivedNegativePositivedNegativePositivedNegative
5Staphylococcus aureusStaphylococcus aureus (GC, 98.4%; UKM, 2,182,976)Staphylococcus aureus (GC, 98.3%; UKM, 2,284,188)Staphylococcus aureus (GC, 42.4%; UKM, 883,874)Staphylococcus aureus (GC, 96.4%; UKM, 2,214,990)Staphylococcus aureus (GC, 17.6%; UKM, 426,194)Staphylococcus aureus (GC, 96.4%; UKM, 2,241,949)
6Pseudomonas aeruginosaPseudomonas aeruginosa (GC, 22.4%; UKM, 23,386)Pseudomonas aeruginosa (GC, 7.4%; UKM, 516,222)Pseudomonas aeruginosa (GC, 8.0%; UKM, 35,293)PositivefPseudomonas aeruginosa (GC, 37.6%; UKM, 20,810), Staphylococcus epidermidis, Micrococcus luteusPseudomonas aeruginosa (GC, 13.7%; UKM, 926,777)
7Cryptococcus neoformansNegativeNegativeNegativeNegativeDermacoccus nishinomiyaensis, Deinococcus geothermalisNegative
10JC polyomavirusJC polyomavirus (GC, 87.2%; UKM, 4,059)JC polyomavirus (GC, 96.8%; UKM, 12,006)JC polyomavirus (GC, 96.7%; UKM, 5,063)JC polyomavirus (GC, 96.8%; UKM, 11,766)JC polyomavirus (GC, 92.7%; UKM, 4,471)JC polyomavirus (GC, 96.8%; UKM, 12,041)
  • a BB, samples were processed with a bead-beating step on the FastPrep instrument; DNAamp, a whole-genome amplification step was performed prior to DNASeq; TTV, torque teno virus; UKM, unique kmer; GC, genomic coverage; human herpesvirus 3, varicella-zoster virus. Entries in bold are the expected result that met the initial positive cutoff of ≥10 times the RPM of the negative extraction control (NEC).

  • b Subsamples K (neat, BB, DNA with no amplification) for samples 2 (Cryptococcus neoformans/C. gattii) and 5 (S. aureus) were the only two samples that met the ≥10 times the RPM of the NEC positivity threshold. However, both subsamples K and H for samples 1, 2, 3, 5, 6, and 10 were positive by secondary analysis using the unique kmer approach.

  • c Subsample H (supernatant, BB, DNA with no amplification), the only positive, was sample 5 for S. aureus by the ≥10 times the RPM of the NEC positivity threshold.

  • d The sample did not initially meet the positive cutoff. However, following the unique kmer analysis approach, the clinical sample was interpreted as positive for P. aeruginosa. Unique kmer and genome coverage ranged by subsamples from 1,039 to 27,333 and 0.01 to 7.4%, respectively. See Fig. 2 for further details.

  • e Following the method comparison study, host depletion studies were performed. The sample processed with saponin/DNase yielded a positive result, with 43,905 enterovirus reads covering 14.9% of the genome.

  • f The sample did not meet the initial positive threshold but was found to be positive using the UKM (top hit) and genomic coverage (8%) approach.