Bacterial Whole Genome Sequencing

Team Members :

1. Himel Sultana
2. Nasren Fatema Jaren
3. Tirtha Debnath
The Team
Contents-
• Sample collection
• Identificaton
• Serotype Identification
• DNA Purification and Extraction
• Polymerase Chain Reaction
• Agarose gel preparation and gel loading
• Imaging by Bio Rad Gel doc XR+ Imager and Band
analysis
• Slide agglutination test
• Next-generation sequencing (NGS)
• Illumina Sequencing Workflow
• DNA library preparation
• Sequencing in iSeq 100
• Data Analysis
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Vitamin A Capsule Campaign Bangladesh Shishu Hospital

Hospital Enrollment

Screening Non-Eligible.(>59 months)

Eligible

If specimen collected Sent to Laboratory

Consent given 2019 IBD Standards

Section 1 & 2 : Screening Form

Enrolled Section 3 : Sample Collection
Section 4 : Investigation
Section 5 : Antibiotic History + Patient Management
Follow up Section 6 : Outcome + Final Diagnostic
Section 7 : Vaccination
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Sample Collection

Blood Agar Plate

Culture on Chocolate Agar Plate

MacConkey Agar Plate

Blood & CSF Sample Collection

Blood Agar Plate showed colonies surrounded by a greenish halo zone referred to as Alpha-
Hemolysis.
 Chocolate Agar Plate showed whitish, round, medium size colonies.
MacConkey Agar Plate showed no colonies.

Culture Plates With Bacterial Growth

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Identification

 Gram staining revealed Gram positive, Lancet shape, Diplococci Bacteria.

 Cytology test performed to count WBC & observe Bacterial infection.
 ICT Test used to detect C polysaccharide cell wall protein of S. Pneumoniae. Two red colored
line on the card indicated positive result.

Gram Staining Cytology Test ICT Test

If more than 9 cells are found in cytology test; ICT test is performed.

When ICT Test card shows positive result but plates do not show any
colony that means patient was previously antibiotic ingested; after then
we perform Antibiotic Assay Test.

Some blank disc with CSF sample were placed onto Blood agar plate
with lawn of Micrococcus sp. Zone of incubation indicated positive
result.
Antibiotic Assay Test
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We perform smart striking because we do not obtain

good colonies in normal culture.

Smart Striking

Optochin is a chemical, ethylhydrocupreine

hydrochloride. Our test Organism were optochin
susceptible because growth of bacteria showed a
zone of inhibiton around optochin disc.

Optochin Test
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Catalase test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies
hydrogen peroxide by breaking it into water and oxygen. The bubbles resulting from production of
oxygen gas clearly indicate positive result.
Our test organism was catalase negative and did not produce bubbles.

Bile solubility test is the ability of bacterial cell to lyse in the presence of bile salts (sodium
deoxycholate).
Our test organism lysed in presence of bile.

Catalase Test Bile Solubility Test

 Our Presumptive Pathogenic Bacteria is Streptococcus pneumoniae

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 What serotype is the

baby infected against?
 The pathogenic gene of
pneumoniae has been
specifically and properly
expressed in its body
Or didn’t happen?
 The baby was
vaccinated but still
infected, Why??
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Serotype Identification
Serotyping

Genotype Phenotype

Pathogenic gene presence Antigen presence

PCR and Gel electrophoresis Slide agglutination test

Positive result Positive result

Serotype(4) identified which was covered by PCV-10

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DNA Purification And
Extraction

 Add 20µl of Qiagen protease in to a 1.5ml micro-centrifuge tube

 Add 200µl of whole blood into the same 1.5ml micro-centrifuge tube
 Add 200µl of buffer (AL) and mix by pulse vortexing for 15 seconds
 Incubate the solution in a water bath of 56 ºC for 10 minutes
 Then briefly centrifuge the tube to remove drops from the inside of the lid
 Add 200µl of ethanol (100%) to the sample and mix again by pulse vortexing for 15 seconds. Then briefly centrifuge
the mixure
 Then transfer the mixture carefully into a QIAamp mini spin column (in a 2 ml collection tube) without wetting the rim
 Close the cap and centrifuge the tube at 8000rpm for 1 minute
 Then place the QIAamp mini spin column in a clean 2 ml collection tube and discard the tube containing the filtrate
 Add 500µl of buffer (AW1) without wetting the rim and centrifuge at 8000 rpm for 1 minute after closing the cap
 Then place the QIAamp mini spin column in a clean 2ml collection tube and discard the tube containing the filtrate
 Add 500µl of buffer (AW2) without wetting the rim and centrifuge at 14,000 rpm for 3 minutes
 Then place the spin column in a new 2ml collection tube and discard the collection tube with the filtrate
 Centrifuge the spin column with the collection tube again at 14,000rpm for 1 minute
 Place the QIAamp mini spin column in a clean 1.5ml micro-centrifuge tube and discard the collection tube containing
the filtrate
 Then add 200µl of elusion buffer (AE) and incubate at room temperature for 1 minute. Then centrifuge the mixture at
8000rpm for 1 minute
 Discard the QIAamp mini spin column and store the micro-centrifuge tube containing the eluted DNA at -80°
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PCR(Polymerase Chain
Reaction)
PCR or Polymerase Chain Reaction is a technique used in
molecular biology to create several copies of a certain DNA
segment.

This technique was developed in 1983 by Kary Mullis, an

American biochemist.

Components of PCR
Components of PCR constitutes the following:
1.DNA Template– The DNA of interest from the sample.
2.DNA Polymerase– Taq Polymerase is used. It is thermostable and
does not denature at very high temperatures.
3.Oligonucleotide Primers- These are the short stretches of single-
stranded DNA complementary to the 3’ ends of sense and anti-sense
strands.
4.Deoxyribonucleotide triphosphate– These provide energy for
polymerization and are the building blocks for the synthesis of DNA.
These are single units of bases.
5.Buffer System– Magnesium and Potassium provide optimum
conditions for DNA denaturation and renaturation. It is also important
for fidelity, polymerase activity, and stability.

Continued……..
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The total PCR procedure was segregated intro three portions:

 Master Mix Preparation

 Template Addition
 PCR Reaction

Master Mix Preparation

 A PCR master mix, sometimes known as super mix or ready mix, is a batch
mixture of PCR reagents at optimal concentrations that can be prepared and
divided among many PCR tubes or 96-well PCR plates.

 The master mix usually includes Taq DNA polymerase, dNTPs, MgCl2 and
buffer.

Template Addition Fig: Master mix preparation

and Template addition
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PCR reaction in Thermocycler

What is Thermocycler?

Thermocyclers, or polymerase chain reaction (PCR) machines, are laboratory instruments that are
used to amplify DNA and RNA samples by the PCR by regulating temperature during cyclical
programs.

Fig: PCR reaction in Thermocycler

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Agarose Gel Preparation and Gel Loading

 Measure 1 g of agarose

 Mix agarose powder with 100 mL 1xTAE in a microwavable

flask

 Microwave for 1-3 min until the agarose is completely

dissolved

 Let agarose solution cool down

 Add ethidium bromide (EtBr) to a final concentration of

approximately 0.2-0.5 μg/mL

 Pour the agarose into a gel tray with the well comb in place

 Place newly poured gel at 4 °C for 10-15 mins OR let sit at

room temperature for 20-30 mins, until it has completely
solidified.
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Agarose Gel Electrophoresis

 Add loading buffer to each of your DNA samples.

 Once solidified, place the agarose gel into the gel box
(electrophoresis unit)

 Fill gel box with 1xTAE (or TBE) until the gel is covered

 Carefully load a molecular weight ladder into the first lane of

the gel

 Carefully load our samples into the additional wells of the gel

 Run the gel at 80-150 V until the dye line is approximately 75-
80% of the way down the gel

 Turn OFF power, disconnect the electrodes from the power

source, and then carefully remove the gel from the gel box

 Using Bio Rad Gel doc XR device that has UV light, visualize
our DNA fragments. The fragments of DNA are usually
referred to as ‘bands’ due to their appearance on the gel
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Imaging by Bio Rad Gel doc XR+ Imager and Band analysis

 The Gel Doc XR+ System is based on CCD high-

resolution, high-sensitivity detection technology
and modular options to accommodate a wide
range of samples

 Here we used 100kb ladder and at the end of gel

running, when we visualized them under the Gel
Doc XR system, we found DNA band at the
position of near about 500bp
 Slide agglutination test

 Basic type of agglutination reaction that is performed on a slide.

 In this method suspension of unknown antigen is kept on slide and a drop
of standardized antiserum is added or vice versa.
 A positive reaction is indicated by formation of visible clumps. E.g. Widal
test, RPR test.

Procedure:
 Took a sterile glass slide
 Add patient specimen which contain specific antigen or not
 Then added specific antiserum against specific serotype of S.pneumoniae
 If specific serotype presence in patient specimen, agglutination can be
occurred and clumping occurred between specific antigen and antibody
 If patient sample doesn’t contain specific antigen on S.pneumoniae thus
don’t occur any agglutination reaction

Result:
We found agglutination and thus we claimed our patient might be infected
with S.pneumoniae serotype 4
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Are the pathogenic genes of S.pneumoniae become mutated?

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Next-generation sequencing (NGS)

Sequencing by synthesis, where millions of small fragments of DNA

are simultaneously sequenced in a massively parallel manner.

Why NGS sequencing by Illumina

• High throughput
• Cost-effective
• Low Error rate
• Identify novel pathogens

Quantify on Qubit and Normalization

 Assess DNA purity  DNA quantify

Nanodrop Qubit
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Illumina Sequencing Workflow

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Workflow of DNA library preparation

1.Fragmentation

2.Adaptor Ligation

3.SPRI Bead Clean-up+ Size

Selection
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4. USER Digestion and Barcoding

With Adaptor
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5 &6. SPRI Bead Cleaning Up and Size Selection (0.8x + 0.75x)

Nucleic Acids Bind Pull Down Separate Wash Separate Elute

with SPRI Beads

Final library for each sample

7. Loading on iSeq 100

Final libraries are quantified and

pooled.Then it was brought to a
concentration of 100 pM in 20microlitre

Also,1.5% of 100pM Phi X is added to final

library.
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8. Sequencing in iSeq 100
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9.Data Analysis

Pairwise
Sequence
Getting From NCBI Reference Alignment by
Reads Genome Collection Blast

FastQC Short Read

Alignment
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Acknowledgement
Thank You