As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves

As you sow so shall you reap

sample to sequencer process

Saria Otani, MRes., MSc., PhD

EURL-AR training course

Denmark, 2019

DTU Food, Technical University of Denmark

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The dress colour “enigma”

e.g., the light exposure, the observer, the device.




(Thompson., et al., 2015)

e.g., the light exposure, the observer, the device (ID’ing).



What do we want to achieve?

Bacterial profiling, isolates or metagenomes.

Functional analyses: gene identifications, AMR,

virulence, diet-decomposition, immunity-related, evolutionary traits.

From sample to sequencer



A project workflow, WGS or metagenome

Plan ahead: what exactly are you looking for? design the experiment

accordingly!

Sample.

DNA extraction.

Library preparation.

Sequencing platforms.

Analyses.





accordingly!

Sample.

DNA extraction.


Sequencing.

Analyses. 1

2

3

4





accordingly!

Sample.

DNA extraction.



Analyses.



Sequencing technology selection

Many classifications for the different methods:


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This is commonly known

as NGS




This is commonly known

as NGS





A bit confusing!

What do I want it for? Gene detection (AMR)? Plasmids?

Evolutionary analyses? Microbiome?




• Short read technologies

– Illumina (MiSeq, NextSeq, HiSeq, NovaSeq…)

– Ion Torrent

– 454

• Long read technologies

– Pacific Biosciences (PacBio)

– Oxford Nanopore Technologies (MinION)




• Short read technologies

– Illumina (MiSeq, NextSeq, HiSeq, NovaSeq…):

Average 300 bp reads

Good accuracy

Error rate ~0.1%

– Ion Torrent: Was the fastest runtime and work-flow in this category (until 2018)

Average 400 bp reads

Error rate ~1%

– 454: Out of market




• Long read technologies

– Pacific Biosciences (PacBio):

Long reads. (Max: 50 kb. Avg.: 10-15 kb)

Low throughput

– Oxford Nanopore Technologies (MinION):

Very long reads (up to 900 kb.)

Fast turnaround time (down to 1.5 hrs)

Portable and real-time sequencing

Large error rates (3-8%)



16

Oxford Nanopore Technologies (MinION):

Long and ultra-long reads – Best high throughput available (100 Gbp) –

Portable, fast and real-time sequencing.



Illumina:

Nanopore:

DNA concentration ≃ 1 ng

DNA concentration > 1000 ng

Comparison example, differences in DNA input between 2

sequencing technologies:





accordingly!

Sample.

DNA extraction.



Analyses.





accordingly!

Sample.

DNA extraction.



Analyses.


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DNA library preparation:

Collection of steps to

prepare DNA to be

read by a sequencing

machine.





prepare DNA to be


machine.

This example is for

Illumina machines.


Add or change Presentation Title or Date via ”Insert”; ”Header & Footer” Oxford Nanopore Technologies, 2019



prepare DNA to be


machine.

This example is for

Oxford Nanopore machines.

Same steps, yet different

ingredients and handling!

Clean up – magnetic beads

Clean up – magnetic beads



DNA library preparation Illumina vs MinIon:

Same steps, yet different ingredients and handling!

Illumina MinIon

Clean up –

magnetic beads

handling the

DNA





accordingly!

Sample.

DNA extraction.



Analyses.





accordingly!

Sample.

DNA extraction.



Analyses.





accordingly!

Sample.

DNA extraction.



Analyses.



DNA extraction

Must know:

The starting material.

The downstream application.



DNA extraction

Starting material

Modified from Printerest.com



DNA extraction

Starting material

Cell lysis


Left after the lysis:

DNA, RNA, protein



DNA extraction

Starting material

Cell lysis

Protein precipitation

Clean up




DNA extraction, what could be different for long reads!

Starting material

Cell lysis

Protein precipitation

Clean up


Different for long reads

Different for long reads



DNA extraction, examples:

1- Lysis.

2- Precipitation.

3- Clean up.







DNA extraction

Quality/integrity of DNA

•Qubit

•Nanodrop

•Bioanalyzer



DNA extraction

A B C D E Ladder



DNA extraction

Quality/integrity of DNA

•Qubit

•Nanodrop

•Bioanalyzer

Why is it important to check

DNA quality?




The dress colour “enigma”

e.g., the light exposure, the observer, the device.



Illumina seq. example:

Sensitivity of AMR detection in microbiomes.

As you sow so shall you reap, act 1

Nanopore seq. example:

Plasmidome characterisation.




Illumina seq. example:

Sensitivity of AMR detection in microbiomes.


Poultry faeces Pig faeces

1:1000

1:100

1:10

1:1

1:0

0:1

10:1

100:1

1000:1

volume volume




Stepwise pattern of AMR profiles

follows the gradual mix of the two

microbiomes.




Plasmidome characterisation.




DNA QC is a key for Nanopore sequencing.

Pure - high concentration - long fragments DNA.

~ 10 Kbp ~ 20 Kbp

Nanopore seq. example: Plasmidome characterisation.


Magnetic beads

(e.g., ZymoBIOMICS)

Modified for increased yield

Gravity flow colums

(e.g., Qiagen Genomic tips)



Sewage Plasmid DNA

> 10 plasmid extraction

protocols were tested

To select and enrich for circular plasmids:

• Linear DNA was digested

• Rolling circle enrichment


Plasmidome characterisation. Pre-sequencing treatment:




Plasmidome characterisation

Comparing plasmidome

throughputs



Comparing plasmidome

throughputs

Plasmidome characterisation

> 4000 contigs

> 2000 potenial circular plasmids




Nanopore from complex samples

Very dependent on DNA and library preparation.

Easy from single isolates





accordingly!

Sample.

DNA extraction.



Analyses. 1

2

3

4

To remember



To remember

Choose your sequencing technology that serves your analysis best (e.g.,

gene detection, identification).

No commercially available kit is optimal for all DNA extraction or

library prep.

A method that works for all, yes.



We have come a long way

Next-Generation sequencing as a diagnostic tool

(Microbiology)



Thank you

Twitter: @SariaOtani

LinkedIn: Saria Otani

Email: [email protected]

mailto:[email protected]

Documents

As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves