CRISPRにターットされるヒト腸内メタ ノム配列の網羅的ータ ースの構築

トーゴーの日シンポジウム2021○杉本竜太1 西村瑠佳1,2 井ノ上 逸朗11)情報・システム研究機構国立遺伝学研究所人類遺伝研究室

2)総合研究大学院大学

ベデ

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Licensed under a Creative Commons 表⽰4.0国際ライセンス
国立遺伝学研究所©2021杉本⻯太（バイオサイエンスデータベースセンター）ゲ

12Viruses
The most diverse and numerous genetic entities on Earth

31
About 10 viruses on Earth
Number of sand grains: 1018
Number of bacterial cells: 1030

About 50 viral species infect one mammalian species
About 5 thousands mammalian specie are known; There are about 300 mammalian infecting viral species
Currently, about 7 thousands viral species are recorded in ICTVThis is a portion of entire virome on EarthVirus evolutions and involvement to the cellular life

• Horizontal gene transfer• Viruses possibly transfer genes between species• Evolutional arms race
• Host evolves immune system, and viruses evolve to counter or escape it• Intriguing origin(s) of viruses• Viruses likely have multiple origins, and they are very old

To understand the evolution of viruses, we need diverse viral genomes

Metagenome contains viral genetic informations

AATACCTGCCTGTCACGCAGGGGGTCGCGGGTTCGAGTCC…
CAGTGTAATGTCAAGATAAACTTGGGACAGCTCATTA…
TTATCGAATCCCTCAGCACGGGCCAGGGAAA…

Metagenome is a mixture of all organisms and viruses in the sample

How do we identify viral genomes from metagenome?

Di culties to infer viral genomes

There is no gene shared across all viral lineages

Archaea

Bacteria

Ribosome
Eukaryotes
Cellular life share genes such as ribosome

fiffi

Unlike cellular life, we cannot de ne a marker genefor viral genomes

CRISPR is a prokaryotic adaptive immunityVirus

Origin of spacers (protospacers)

Viral DNA

Innate immunity

CRISPRCRISPR

Restriction

Acquisition
Expression

Spacers+

Host chromosome

Prokaryotic cells

Using CRISPR spacers, we can identify viral genomes from metagenome

Metagenome reads

Detect CRISPRs

Genome assemblyCollect spacers

Contigs

Identify contigs targeted by CRISPR

Applying graph-based method to extract CRISPR targeted sequences
A B
A B

contig1
contig1

CC

D EF
D E F

G

FE D
FE Dcontig2
contig2

CC

B A
B AO

contig3
contig3OK

K L M NL MNPQR

P QR

GH

H

I J

NM L
NM L

S

P QRKK

P QR

contig4

I J

STTUV

contig4A
BFCEOPDSQRKNTULM

VU V

ResultAnalyzed 11,817 human gut metagenome dataset (50.7 Tb)
Assembled 180,068,349 (767.7 Gb) contigs
Detected 11,223 CRISPRs
Extracted 1,969,721 CRISPR spacers

Using them, we detected 11,391 CRISPR targeted sequences
The extracted sequences include• 257 crAssphage• 5 huge phages (> 200 kb)• 766 Microviridae• 56 Inoviridaefi

We also identi ed the hosts of 7,937 (69.7%) CRISPR targeted sequencesphages
ailed phages

Classi cation of discovered CRISPR targeted sequences
391 (15.31%)

116 (4.54%)766 (8.67%)

391 (15.31%)

14
Number of sequences

2,641 (29.89%)

2,047 (80.15%)

16

5,267 (59.60%)

12
10
8

2,047 (80.15%)

6

5,267 (59.60%)

300,000420

100,000

150,000

200,000
Length (bases)

250,000

Large (> 20 kb)

300,000

Caudovirales

Small (< 20 kb; k = 11)

Caudovirales

1.0

Inoviridae

Microviridae

0.6

Microviridae

UnclassifiedPlasmid-like

Small (< (<20 kb)
Small20 kb)

0.6
0.4
0.2

0.4

Plasmid-like

Inoviridae

0.8
Coding ratio

Coding ratio
Caudovirales

Small (< 20 kb) Unclassified
Plasmid-like

Microviridae

Large(> 20 kb)Large (> 20 kb)
1.0

0.8

Unclassified

Small (< 20 kb)

Large(> 20 kb)
Inoviridae

100,000 150,000 200,000 250,000 300,000
Length (bases)

020
kb;kb;
k = 13)k = 11)d-like56 (0.63%)
2,641(29.89%)107 (1.21%)

Tailed phages
Not tailed phages,000 300,00000b

Unclassified

0CaudoviralesPlasmid-like

Unclassified

Caudovirales

Inoviridae

Microviridae

Plasmid-like

Unclassified

0.2
Plasmid-likefi

icroviridae

Unclassified

0Caudovirales

Plasmid-like

Unclassified

Caudovirales

Inoviridae

Microviridae

Plasmid-like

Unclassified

Predicted hostsb55%

NA (2,036)
Firmicutes (3,790)
Ambiguous (1,418)

Euryachaeota (1)Actinobacteria (1,181)

Verrucomicrobia (3)Fusobacteria (10)Proteobacteria (105)cBacteroidetes (2,847)

Number of sequences

1,600

High-GC

Low-GC

a

1,400
1,200
1,000
800
600
400
200
00102030405060GC content (%0.99920.86150.5618

Phylogeny of Microviridae species
679

Fig. 20.99920.8615
0.98

Host phylum
Bacteroidetes

10.99230.7793Firmicutes
Proteobacteria

0.70370.968

1Gokushovirinae Bog1183 53

1

Chimpanzee faeces associated microphage 1
Chimpanzee faeces associated microphage 3

0.543

0.94230.99720.909

10.8469 1
10.53941
10.95720.957

0.95720.9572
0.63130.9572

10.9967

11

1

Marine gokushovirus
Gokushovirinae Bog5712 52
Gokushovirinae Fen7875 21
Gokushovirinae Fen672 31
Gokushovirinae Bog8989 22
Chlamydia phage CPG1
Chlamydia phage 2
Chlamydia virus Chp1
Microviridae Bog1249 12
Microviridae Fen4707 41
Alces alces faeces associated microvirus MP21 4718
Microviridae Fen7940 21

1

Parabacteroides phage YZ-2015b
Parabacteroides phage YZ-2015a
Microviridae Fen685 11
Microviridae Fen7895 21
Microviridae Bog5275 51
Microviridae Fen7786 21
Microviridae Fen418 41
Microviridae Fen2266 11
Microviridae Bog9017 22
Microviridae Fen7918 21
Alces alces faeces associated microvirus MP15 506710.6497

10.6096

10.56420.8845111111111

10.8430.8601
0.96790.9997

Lepus americanus faeces associated microvirus SHP1 6472

1
10.9999
0.96990.9728110.9736
0.999310.9979

0.751

10.5237
0.66140.96220.7532

10.8786
1
1 1

110.9399

1
1

1

10.8269

110.5029

10.9998
10.9985
10.99281

10.9854
0.9767

1

1

11

1

10.9901
0.9887
0.99190.9997110.5531
0.99240.81840.828610.7083
10.9954

10.5298

0.98830.962910.9883
0.976
11

1

11
1
10.5508
1680

1
1

1

10.9997

0.7632

1

Microviridae phi-CA82
Spiroplasma virus SpV4
Alces alces faeces associated microvirus MP10 5560
Alces alces faeces associated microvirus MP12 5423

0.99980.97891

10.79440.87

0.9281

0.984210.8506

0.906

0.90811

11

1

10.85240.841

110.6469
0.56770.5188Chimpanzee faeces associated microphage 2

10.8786

Lynx canadensis associated microvirus CLP 9413

10.9069

1Escherichia phage EC6098
Alces alces faeces associated microvirus MP11 5517
Bdellovibrio phage phiMH2K

0.96930.68240.84181

10.9998
0.99830.8273 0.906
1 0.9998

0.66960.864510.9772

10.83751

1

10.9835

10.5618

0.9992
Microviridae IME-16

Alces alces faeces associated microvirus MP3 6497
Escherichia phage G4Escherichia phageaID21
Escherichia virus phiX174
Escherichia phage St-1
Eel River basin pequenovirus

10.3Summary• Discovered about 10 thousands CRISPR targeted genomes• Substantial portion of them encode capsids• 70% of them were host resolved at phylum level• Discovered many novel small genomes

Next steps• Further inspection of novel genomes• Discover RNA viruses and archaeal viruses• Construct database for viral genomes and proteins