mngs

A metagenomics-based diagnostic approach for central nervous
system infections in hospital acute care setting

Number of suspected CSF specimens: n = 74
While a statistically significant correlation (R = 0.63; p < 0.001) was observed between approximate titers of spiked
pathogens with their respective NT reads, the qPCR C T values for different pathogens were more precisely correlated to
their mNGS NT reads (R = 0.96; p < 0.0001)
Bacterial and viral strains
Escherichia coli ([ATCC] 25922)
Streptococcus pneumoniae (ATCC49619) Blood agar/ chocolate agar
Streptococcus agalactiae (ATCC12386)
Haemophilus influenzae (ATCC10211)
Neisseria meningitidis (ATCC 13090)
37oC, 5% CO2
Herpes Simplex Virus 2 (ATCC VR-540)
Human Adenovirus type 7 (ATCC VR-7)
E. coli, S. pneumoniae and S.
agalactiae
Viral stock
maintaining
Liquid
Nitrogen
Dulbecco’s Modified Eagle’s Medium (DMEM)

supplemented with 10% DMSO at − 80 °C.
5 specimens: spiked
Test optimization and spiking
CSF specimens (n = 9) negative
by standard microbiological 4 specimens: unspiked
methods+ 1 nuclease free
water (NFW)
1 unspiked nuclease free water
Clinical validation mNGS IDseq

74 CSF specimens
Neisseria meningitidis (4.3 x10^8 CFU/mL)
Freshly prepared
in PBS Turbidity = 0.5 MeFarland standard
Bacterial suspensions Herpes Simplex Virus 2 (2.8 x10^5 TCID50/mL)
(diluted 10-,100-,1000-fold)
Human Adenovirus type 7 (2.8 x10^5 TCID50/mL)
Spiking 0.5mL CSF

specimens
Vortex in 10s
• DNA extraction: QIAsymphony instrument (Qiagen)

1ng (Nextera XT DNA Sample Preparation)
• DNA concentration: Qubit 2.0 flurometer/ Qubit dsDNA
HS assay
qPCR and mNGS
IDseq
Spiking method
• Quality Control: Spiked samples can be used to assess the performance and accuracy of laboratory techniques
or analytical methods. By adding a known amount of a substance to a sample, scientists can determine if the
method can accurately detect and quantify the spiked substance.
• Recovery Studies: Spiked samples can be used to assess the recovery efficiency of a sample preparation or
extraction method. Known amounts of a substance are added to a sample before processing, and the recovery
rate is determined by comparing the measured concentration of the spiked substance to its known
concentration.
• For determining the analytical sensitivity of mNGS, they used a large number of bacterial and viral strains
for spiking (-) CSF specimens to ensure that the assay performs reliably and accurately in detecting a broad
range of pathogens in various clinical or research settings.
• For internal control of extraction, they used plasmid instead of bacterial and viral strains because:
 Consistency: Plasmids provide a consistent and reproducible source of DNA for internal control.
 Quantification: Plasmids can be accurately quantified using various methods, such as spectrophotometry or
fluorometry. This allows for precise determination of the concentration of the internal control DNA added
to the samples, enabling better control over the amount of control DNA spiked into each sample.
 Stability: Plasmids are generally more stable than bacterial and viral strains. They can be stored for
extended periods without significant degradation or loss of viability  it provides a reliable reference
throughout experimental process.
 Contamination control: Using plasmids as internal controls helps mitigate the risk of introducing additional
pathogens or contaminants into the samples.
Library normalization
• Purpose: Normalization aims to remove technical biases (differences in RNA or DNA input, library
preparation efficiency, or sequencing depth) to enable meaningful comparisons between samples.
• Impact on Data Analysis: -crucial for accurate downstream analysis, such as differential gene expression
analysis or identification of genomic features. Normalizing the read counts or coverage allows for valid
comparisons of gene expression or genomic features between samples, reducing the influence of technical
biases.
• Types of Normalization:
 Reads per sample normalization: This method adjusts the read counts by scaling them to a common
library size or sequencing depth. The total number of reads for each sample is divided or multiplied by a
scaling factor to achieve a desired target library size.
 Trimmed mean of M-values (TMM): TMM normalization calculates scaling factors based on the
geometric mean of expression ratios for a set of reference genes. This method accounts for differences in
library composition and attempts to mitigate the impact of highly expressed genes.
 Quantile normalization: This method aligns the distributions of read counts or coverage across samples
by matching the quantiles of the distributions. It ensures that the overall distribution of reads is similar
between samples, reducing technical variation.
 DESeq/DESeq2 normalization: These methods use a statistical approach to estimate size factors based on
the assumption that most genes are not differentially expressed. The size factors are then used to adjust
the read counts for each sample.
Laboratory validation of a clinical metagenomic sequencing
assay for pathogen detection in cerebrospinal fluid
The transposase-mediated library preparation method simplifies the library construction

process by combining the fragmentation and adapter ligation steps into a single reaction
 Fragmentation and adapter ligation: DNA samples are subjected to enzymatic fragmentation using
a transposase enzyme. The transposase simultaneously cuts the DNA and inserts sequencing
adapters into the fragments.
Methylation-based enrichment methods: selectively remove methylated host DNA from a sample enriching for
unmethylated pathogen DNA.
 Bisulfite Conversion: widely used method for DNA methylation analysis. It converts unmethylated cytosines to uracils
while leaving methylated cytosines unaffected. Subsequent PCR or sequencing-based analysis can then specifically target
and amplify the unmethylated regions, enriching for pathogen DNA. Kits such as the EZ DNA Methylation Kit (Zymo
Research) or the MethylEdge Bisulfite Conversion System (Promega) can be employed for bisulfite conversion.
Methylation-based enrichment methods
 Methylated DNA Immunoprecipitation

(MeDIP): MeDIP utilizes antibodies
specifically targeting methylated DNA to
immunoprecipitate methylated DNA
fragments. Commercial kits like the
MethylMiner Methylated DNA Enrichment
Kit (Thermo Fisher Scientific) can be
employed for MeDIP.
Methylation-based enrichment methods
 Methyl-CpG Binding Domain (MBD) Protein-

Based Enrichment: MBD proteins bind
specifically to methylated DNA regions.
Methylated DNA fragments can be captured using
MBD protein-based enrichment methods, followed
by the removal of methylated host DNA. The
MethylCap Kit (Diagenode) and the
MethylCollector Ultra Kit (Active Motif) are
examples of kits that utilize MBD proteins for
DNA methylation enrichment.
 Principle: MagneZoom-GSH are silica-
coated superparamagnetic beads covalently
linked to reduced Glutathione for affinity
purification of proteins containing
Glutathione-S-transferase (GST) fusion tag

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A metagenomics-based diagnostic approach for central nervous

system infections in hospital acute care setting

Dulbecco’s Modified Eagle’s Medium (DMEM)

Clinical validation mNGS IDseq

Human Adenovirus type 7 (2.8 x10^5 TCID50/mL)

Spiking 0.5mL CSF

• DNA extraction: QIAsymphony instrument (Qiagen)

qPCR and mNGS

The transposase-mediated library preparation method simplifies the library construction

 Methylated DNA Immunoprecipitation

 Methyl-CpG Binding Domain (MBD) Protein-

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