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mNGS

metagenomic Next-Generation Sequencing

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The Team

Cristina Tato,

Director

Rapid Response (RR) group

Vida Ahyong,

Scientist

RR group

Maira Phelps,

Clinical Research

Coordinator

RR group

Manu Vanaerschot,

Scientist

RR group

Katrina Kalantar,

Computational

Biologist

Liz Fahsbender,

IDseq

Application

Scientist

Olivia Holmes

IDseq

Product

manager

Michelle Tan,

Scientist,

Sequencing group

Angela Detweiler,

Scientist,

Sequencing group

Lusajo Mwakibete,

Research Associate

Patrick Ayscue

Sr. Biosecurity fellow

RR group

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Introduction

Pathogen landscape

NGS toolbox for infectious diseases

Sample landscape

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Pathogen landscape

Organism	Viruses	Bacteria	Eukaryotic microbes (yeast, amoebas, worms, etc)
Genome composition	ssRNA, ssDNA, dsDNA, or dsRNA	dsDNA	dsDNA
rRNA	None, uses host ribosomes	16S RNA	18S RNA
Genome size	2 Kb - 1 Mb	1 Mb- 5 Mb	200 Kb - 100,000 Mb

In terms of pathogens, in the order of small to large, we have viruses, bacteria and then eukaryotic microbes. When trying to sequence any of these known or unknown pathogens, there are a few issues to keep in mind which will influence detection, identification, and surveillance. First, genome composition, while bacteria and eukaryotic microbes contain a DNA genome, some viruses only have RNA genomes. Therefore when using sequencing to identify unknown pathogens that could be viral, it is best to perform RNA sequencing rather than DNA sequencing. When performing RNA sequencing, we know that most of the RNA sequencing will be from ribosomal RNA (rRNA) rather than messenger RNA. One advantage is that rRNA can help type the bacteria (such as 16S seq methods) however if your sample has a lot of host background, you will mostly sequence host rRNA.

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Sample type landscape

Cerebral Spinal Fluid

No infection: 0% microbial

With infection: <1% - 5%

Rectal or Vaginal swabs or stool

50% microbial (mostly bacteria)

Nasal or oral swab

50% microbial (mostly bacteria)

Host

Microbial

Whole Blood: lot of background

No infection:

During infection: <<<0% microbial

Serum or Plasma

10-100x less host material

Tracheal Aspirate

5% microbial, 95% human

Different sample types have different RNA contents!

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NGS toolbox for infectious diseases

	Whole genome & transcriptome sequencing	Amplicon sequencing	16S & 18S rRNA sequencing	Metagenomic (shotgun) sequencing
Starting material	DNA/RNA from (cultured) isolate	DNA/RNA from a sample	DNA/RNA from a sample	DNA/RNA from a sample
Primers used to amplify sequences	Random primers	Organism-specific primers	16S/18S-specific primers	Random primers
What is sequenced?	Organism of interest only	Organism of interest	rRNA from all bacteria, fungi and eukaryotic pathogens in sample	All DNA/RNA from all organisms in sample
Resulting data	DNA sequence or gene expression of target organism	RNA/DNA sequence of targeted organism	rRNA sequences of all bacteria, fungi and eukaryotic pathogens	All DNA/sequences from all organisms in sample

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NGS toolbox for infectious diseases

	Whole genome & transcriptome sequencing	Amplicon sequencing	16S & 18S rRNA sequencing	Metagenomic (shotgun) sequencing
Starting material	DNA/RNA from (cultured) isolate	DNA/RNA from a sample	DNA/RNA from a sample	DNA/RNA from a sample
Primers used to amplify sequences	Random primers	Organism-specific primers	16S/18S-specific primers	Random primers
What is sequenced?	Organism of interest only	Organism of interest	rRNA from all bacteria, fungi and eukaryotic pathogens in sample	All DNA/RNA from all organisms in sample
Resulting data	DNA sequence or gene expression of target organism	RNA/DNA sequence of targeted organism	rRNA sequences of all bacteria, fungi and eukaryotic pathogens	All DNA/sequences from all organisms in sample

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NGS toolbox for infectious diseases

	Whole genome & transcriptome sequencing	Amplicon sequencing	16S & 18S rRNA sequencing	Metagenomic (shotgun) sequencing
Starting material	DNA/RNA from (cultured) isolate	DNA/RNA from a sample	DNA/RNA from a sample	DNA/RNA from a sample
Primers used to amplify sequences	Random primers	Organism-specific primers	16S/18S-specific primers	Random primers
What is sequenced?	Organism of interest only	Organism of interest	rRNA from bacteria, fungi and eukaryotic pathogens in sample	All DNA/RNA from all organisms in sample
Resulting data	DNA sequence or gene expression of target organism	RNA/DNA sequence of targeted organism	rRNA sequences of bacteria, fungi and eukaryotic pathogens	All DNA/sequences from all organisms in sample

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NGS toolbox for infectious diseases

	Whole genome & transcriptome sequencing	Amplicon sequencing	16S & 18S rRNA sequencing	Metagenomic (shotgun) sequencing
Starting material	DNA/RNA from (cultured) isolate	DNA/RNA from a sample	DNA/RNA from a sample	DNA/RNA from a sample
Primers used to amplify sequences	Random primers	Organism-specific primers	16S/18S-specific primers	Random primers
What is sequenced?	Organism of interest only	Organism of interest	rRNA from bacteria, fungi and eukaryotic pathogens in sample	All DNA/RNA from all organisms in sample
Resulting data	DNA sequence or gene expression of target organism	RNA/DNA sequence of targeted organism	rRNA sequences of bacteria, fungi and eukaryotic pathogens	All DNA/RNA sequences from all organisms in sample

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SARS-CoV-2 (SC2) sequencing

Amplicon sequencing

(e.g. ARTIC protocol)

Reverse transcription using random primers

PCR of SC2 sequences using SC2-specific primers

DNA library prep

Metagenomic sequencing

Reverse transcription using random primers

RNA library prep

Metagenomic sequencing with spiked primer enrichment

Reverse transcription using random primers + SC2-specific primers

RNA library prep

SC2 whole genome sequencing
High risk of cross-contamination!

Detection of SC2
Detection of co-infections
SC2 whole genome in case of very high viral titers

SC2 whole genome sequencing
Detection of co-infections
Lower risk of cross-contamination compared to amplicon sequencing

MSSPE

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Metagenomic next generation sequencing

Comprehensive analysis of all of the genetic material present in a sample

RNA or DNA mNGS? That depends on your pathogen(s) of interest...

mNGS

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Use cases for mNGS

Outbreak detection and prevention

Identify a novel pathogen

Identify coinfections

Characterize the microbial landscape

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Use case: Saha et al., mBio, 2019

mNGS to identify etiologies of pediatric meningitis in Bangladesh

Known Positives

Known Negatives

Unknown

Water Controls

Microbial Culture, PCR, Serology

No pathogen identified by standard testing

No pathogen identified; symptoms resolved quickly

Collect Patient CSF Samples

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mNGS pipeline

Study design

xNA extraction

Library prep

Sequencing

Data analysis

Type(s)
Controls
Storage
Lab setup

Type(s)
Controls
QC

Host removal
Fragmentation
dsDNA generation
End-repair
Adapter ligation
Barcoding & PCR
Clean-up
Pooling
QC

QC data
Host removal
Alignment
Blast
Ranking of hits

Machine
Read length
Loading conc.

Pathogen ID

(1-2 days)

(1-2 hrs)

(0.5-3 days)

(0.5-4 hrs)

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Lab setup

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Lab setup

Follow institutional guidelines for biosafety containment

Biosafety level 1 - 2 - … (BSL-1, BSL-2, …)
Note: Shield inactivates dangerous pathogens -> Biosafety level 1

Avoid dust and high humidity levels

Preventative measures to avoid PCR amplicon contamination at all cost

Physical separation of Pre-PCR and Post-PCR
Extra lab precautions (see next slides)

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Lab setup

No PCR allowed
xNA extractions
Lib prep up to setting up the USER/Q5 mastermix (no PCR)

No PCR allowed
xNA extractions of infectious samples

BSL-2 Pre-PCR

BSL-1 Pre-PCR

BSL-1 Post-PCR

Lib prep from barcoding PCR
Lib QC
Lib amplification if needed
Sequencing

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Lab setup

Extra lab precautions:

Sticky floor pads at the entrance of Pre-PCR rooms, changed weekly
Ice buckets color coded to the lab spaces - either Pre-PCR or Post-PCR only - no mixing
Rotation of index primers so the same indices are never used in the same month
Reagents like H₂O, SPRI beads, DNAse, Proteinase K, etc are aliquoted into single-use tubes
Pipets inside the PCR hoods are never taken out. All hoods are sprayed down before and after use with 70% ethanol and RNAseZap
Reagents are not shared between the different rooms
PCR products never enter the Pre-PCR rooms nor samples with high nucleic acid quantities (i.e.: stool, tissues, bacterial cultures, bacterial colonies)

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Lab setup

Other considerations:

Power: Uninterruptible power supply (UPS) or surge protector
Internet connection
Environmental conditions, e.g. for iSeq:

Dust issues: Pollution degree rating of II (non-conductive pollution only, rare conductive pollution)
Ventilation: Up to 2048 BTU/hr @ 600W. Buy extra air filters for the iSeq.
Humidity: Non-condensing 20-80% Relative Humidity
Temperature: 15°C to 30°C (22.5°C ± 7.5°C)

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Lab setup

Lab procedures by location

BSL2 Pre-PCR Biosafety Cabinet

Sample inactivation in DNA/RNA Shield
Nucleic acid extraction (if sample is not deactivated)

BLS2-Pre-PCR Room

Storage of original samples at -80C

BSL1 Pre-PCR PCR hood

Nucleic acid extraction (if sample is deactivated)
Nucleic acid quantification/quality control:

Aliquots can be made in Pre-PCR and run in Post-PCR

DNA/RNA library prep: Fragmentation to setting up the USER/Q5 master mix

BSL1 Post-PCR PCR hood

Post-PCR SPRI clean-up

BSL1 Post-PCR Bench

PCR reaction using USER/Q5 master mix
Library quantification & quality control
Library pooling
qPCR Machine for additional library amplification
Illumina sequencing
Storage of NGS libraries -20C

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Next: mNGS study design, lab setup and sample preparation