Mass sequencing of the faecal

virome: a study in children with

early onset of islet autoimmunity
Lenka Kramn1, Kateina Holkov1, Sami Oikarinen2, JuhaPekka Pursiheimo3, Mikael Knip4, Olli Simell5, Riitta Veijola6,
Jorma Ilonen7, Heikki Hyty2, Ondej Cinek1
1Charles University in Prague, Czech Republic, 2University of Tampere, Finland, 3University of Turku,
Finland, 4University of Helsinki, Finland, 5Turku University Central Hospital, Finland, 6University of Oulu,
Finland, 7University of Turku, Finland

Presenter: Lenka Kramn

Disclosed no conflict of interest

Hypothesis
Viruses are linked to type 1 diabetes

Virus studies

Candidate virus
approach:
specific probes

Mass sequencing:
unbiased approach
- new virus discovery,
- yet only 1 study of
virome sequencing in
diabetes

Objectives
to sequence all viruses in the gut before islet
autoimmunity onset:
= primary
replication
Any association with autoimmunity
onset?

site for many viruses

important
to get the whole sequences of =the
most immune
organ
abundant entities

to compare the next-generation sequencing and

specific PCR: sensitivity, specificity, crosscontamination control

Study subjects
Finnish DIPP (Diabetes Prediction and Prevention)
cohort
19 patients: evenually progressed to T1D
19 controls: matched for HLA risk, gender, date
and place of birth
Altogether 96 stool samples
-9 months -6 months -3 months AI onset

patient
control

T1D

Method
stool sample:

contains the rests of

food, bacteria,
viruses
-centrifugation
- filtration (0.45m)
-ultracentrifugation
(80000xg, 10C, 3h)
- DNase + RNase
treatment

RNA + DNA
isolation

Method
Reverse
transcription

T1
RNA / DNA
NNNNNNNN

T1

Second strand
synthesis

T1
NNNNNNNN

NNNNNNNN

cDNA

Amplification
with the tags
NNNNNNNN
NNNNNNNN
NNNNNNNN

T1

T1

NNNNNNNN
NNNNNNNN
NNNNNNNN

NNNNNNNN
NNNNNNNN
NNNNNNNN
NNNNNNNN
NNNNNNNN
NNNNNNNN

dsDNA

T1

T1

Method
Sample 1 Sample 2 Ladder

Next-generation
sequencing

Nextera XT
MiSeq Illumina
24 samples / run
app. 15 mil.
sequences per run

Results:
The overall sequence content
Viruses
2%
Bacteria

Total sequences
per sample:
200 000
1 500 000

14%

10%

Virus sequences
per sample:
4 000 30 000

Eukaryota

74%

No hit

Results
NGS findings: human viruses
Virus

Cases
(n=48)

Controls
(n=48)

Association:
OR [95% Cl]

Enterovirus
Parechovirus
Bocavirus
Sapovirus
Anellovirus

0
3
1
1
1

1
2
2
0
1

1.5[0.25 8.9]
0.5[0.04 5.5]
-

-3months

10
-

7
1

3
-

1670
2
2
-

Matching
pair ID
1.5
2.3.4.5.
10
6.703
7.
3041 8.3
1
7
9.49
10.
6
1
11.8
39924
12.2291
13.
14.15.16.11
1
17.
1
18.19.1

control

case

control

4
-

case

control

Parechovirus

case

Results:
In detail

19
3
1
-

-6 months

10
703
3
1
49
7
1
39924
2291
11
1
1
-

control

case

Matching
pair ID
1.
5
2. 3. 4
4. 5.
1
6.1
7.
10
3041
8.7
9.1
10.
1670
6
11.
8
12.2
3
13.14.2
15.16.17.1
18.
19.

control

case

control

Parechovirus

case

Results:
In detail

-3 months

19
3
1
-

-9 months

10
-

7
1

3041
-

3
-

1670
2
2
-

6
8
1
-

10
703
3
1
49
7
1
39924
2291
11
1
1
-

control

control

4
-

5
-

-3 months
case

case

control

Parechovirus

Matching
pair ID
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.

case

Results:
In detail

-6 months

19
3
1
-

Results:
The whole virus genomes assembly
Human enterovirus: Echovirus 25,
124 reads

Results:
PCR confirmation of NGS findings
Virus

Cases
(n=48)

Enterovirus
Parechovirus
Bocavirus
Sapovirus

7
3
2
1

Controls
(n=48)

Association:
OR [Cl 95%]

3
2.5[0.61 10.2]
8
0.35[0.09 1.4]
2 NGS detect viruses
in
l
0 quantity 100 copies/
of isolated NA and
higher

Results: Bacteriophages findings

Phage

Positive cases

Positive controls

Islet autoimmunity

n(%) samples of 48 n(%) samples of 48 association: OR [95% CI]

Any bacteriophage

27 (56%)

25 (52%)

1.18 [0.53 2.6]

Escherichia phage

9 (18.8%)

2 (4.2%)

4.5 [0.97 21]

Lactobacillus phage

5 (10.4%)

2 (4.2%)

4 [0.45 35]

Lactococcus phage

4 (8.3%)

5 (10.4%)

0.75 [0.17 3.3]

Clostridium phage

2 (4.2%)

1 (2.1%)

2.41 [0.17 35]

Enterococcus phage

2 (4.2%)

4 (8.3%)

0.33 [0.03 3.2]

Streptococcus phage

2 (4.2%)

1 (2.1%)

2 [0.18 22]

Bacteroides phage

1 (2.1%)

3 (6.3%)

0.33 [0.03 3.2]

Bifidobacterium phage

1 (2.1%)

4 (8.3%)

0.22 [0.02 2.1]

-9 months

-3 months

456

5
-

2613
5365
1
4

249
12
3
-

2
4152
1268
1
-

1
-

control

control

1
-

2
-

case

case

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.

control

E. coli phage

Matching
pair ID

case

Bacteriophages
clustering

-6 months

3
1
4
358
23935
5
73
77
-

1
86
2
36
-

Conclusion
No association with islet autoimmunity found:
NGS/ PCR
Human viruses / bacteriophages
Method:
NGS is less sensitive than PCR
real virus sequencing, quantitative
information and the whole picture of all viruses
Sequence content:
richness of bacteriophages:
modulation of immune system?
What are those 74% of unknown sequences?

Acknowledgements:
DIPP study steering committee:
Mikael Knip, Olli Simell, Riitta Veijola,
Jorma Ilonen
Funding:
- Ministry of Health of the Czech
Republic (IGA MZ 11465-5)
- the Finnish Centre for International
Mobility (CIMO): Government
Scholarship Pool programme
prof. Heikki Hyty
and the group of Department of
Virology, University of Tampere,
Finland
Ondej Cinek
and a collective of Laboratory of
molecular genetics, Charles
university in Prague

Thank you for attention

NGS and PCR

comparison
Empty circles: sample
has been found positive
only by NGS

Black dots: sample has

been found positive.
Below the grey zone:
only by PCR

Threshold for
positivity at 50
reads

Phage and its bacteria findings

Grey zone:
threshold of
50 reads
Phage and
its bacteria
found within
the sample
Only
bacteria
found

Other viruses
Human viruses of low quantity
(less than 50 reads)

Plant viruses of low quantity

(less than 50 reads)

Norovirus, Rotavirus,
Papillomavirus, Rhinovirus

Tobacco mild green mosaic virus,

Turnip vein-clearing virus,
Pepper mild mottle virus,
Paprika mild mottle virus and
Grapevine Syrah virus

Raw NGS reads after trimming and filtering

Velvet: de-novo assembly

100,000 randomly selected

reads

BLAST:
against whole viral genomes

BLAST:
against whole viral genomes

Iterative algorithm:
compares BLAST results and
identifies the best matches

Identification of viruses
at the genus level

specific PCR:
results confirmation

BWA mapper:
Enterovirus VP1 PopSet
Parechovirus VP1 PopSet
Bocavirus VP1 PopSet

Identification
of the specific viral genera
at the genotype level

Megan5:
content viewer

Qualitative and
quantitative information
of the sequence content:
-virus
-bacteria
-human, animal, plant

All human virus results

1 control: 1%

3 cases: 3.1%
2 controls: 2.1%

control

case

control

case

control

control

case

case

control

-3 mo

Anellovirus
-9 mo
-6 mo -3 mo

case

control

control

case

case

control

-3 mo

1 case: 1%
1 control: 1%

1 case: 1%
Sapovirus
-9 mo -6 mo

case

control

case

control

case

-3 mo

control

Bocavirus
-9 mo -6 mo

case

control

control

case

case

control

case

case

Parechovirus
-9 mo -6 mo

control

Enterovirus
-9 mo -6 mo -3 mo

1 case: 1%
2 controls: 2.1%

17

40

10

703

22

124

15

10

3041

19

9320

19

29

49

29

117

22703

5087

1670

1010

40

48

39924

2291

11

21

53

NGS analysis pipeline

Consenzus

Identification by BAST:
the whole genomes