nbrc genome sequencing projects for 20 years and current ......nbrc genome sequencing projects for...

NBRC genome sequencing projects for 20 years and current services using genome sequence as BRC

Hiroko Kawasaki Ph.D. Director

Industrial Innovation Division, NITE Biological Resource Center (NBRC)

E-mail: [email protected]

Outline

• Organizational Structure of NBRC

• Current Status of NBRC Culture Collection

• Highlighted Activities

– Genome sequencing

– Information services using genome sequencing

• Conclusion

2

On-site inspection

Causal analysis of product accidents

Development of Standards

Development of Analysis Method

Accreditation

Separation, depositary and analysis of Microorganism

Risk Assessment

Consumer Product Safety

Planning and Administration

Chemical Management Conformity Assessment

Mission of National Institute of Technology and Evaluation (NITE)

Biotechnology (NBRC)

Global Center for Evaluation Technology

NITE aim is to provide reliable technologies and information that underpin public safety and help overcome the challenges of the future.

Tohoku Regional Office

NITE Headquarters & Genome analysis laboratories

NITE Biological Resource Center (NBRC)

Patent Depository Centers (NPMD and NITE-IPOD)

Tokyo Kazusa

Sendai

Location of NBRC

Osaka

Global Center for Evaluation Technology & Kansai Biotechnology Office

NITE Long-term Preservation Center

Genome Information

Culture Collection

International

Cooperation

Biosafety Informatio

n

Patent Depositar

y

Industrial Support

& Inspection

Activities of NBRC

Preservation/Distribution of Biological Resources

Supporting Access and Sustainable Use of Biological Resources

Provision of Useful Information on Biological Resources

• NBRC CC strains: approx. 32K strains ◆ Microbes mainly deposited from Japanese or foreign researchers

◆ Managed under ISO9001

◆ Identified to species level

◆ Usable of online catalogue

◆ Fee: 8,000 JPY

• NBRC R&D(RD) strains: approx. 60K strains ◆ Microbes collected by NBRC staff and collaborators

◆ Classified to genus level (not identified)

◆ Custom made supply

(Domestic only)

◆ Fee: 500-1,000 JPY/year

(Domestic only)

Unique services of NBRC culture collection

NBRC Culture Collection strains

(As of March 31, 2018)

Filamentous

fungi

37%

Bacteria

33%

Yeast

15%

Actinomycetes

12%

Microalgae

2%

Archaea

1% Bacteriophages

0.4%

■Filamentous fungi 11,906

■Bacteria 10,450

■Yeasts 4,768

■Actinomycetes 3,885

■Microalgae 705

■Archaea 335

■Bacteriophages 117

Total Holdings 32,166

■Human cDNA clones 55,399

■Human GatewayTM

Entry Clones 43,567

■Domestic Industry 3,584

■Domestic Academy 3,158

■Foreign Industry & Academy 499

■Domestic other 115

■Foreign Exchange 54

■Foreign other 23

■Domestic Exchange 7

Number of strains distributed 7,440

NBRC CC strains Distribution

Price (¥)/strain

Domestic

Industry 8,000

Academic 4,000

Foreign

Industry & Academy 8,000

Domestic

Industry

48%

Domestic

Academy

42%

Foreign

Industry &

Academy

7%

Domestic

other

2%

Foreign

Exchange

1%

Foreign

other

0.31% Domestic

Exchange

0.09%

(As of March 31, 2018)

Distribution from NBRC Culture Collection

0

2,000

4,000

6,000

8,000

10,000

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017

1,603 2,225 2,136 2,199 2,446 2,465 2,411

2,796 2,780 2,919 2,913 2,822

2,628 2,648 2,600 2,620

5,518

6,538 6,144

7,213

8,019

7,363 7,798

8,819 9,354

9,149 8,824

8,378

7,800

9,778

9,060

7,584

Order

Resources

Total 7,584 = 7,440 (microbes) + 144 (DNA resources)

Total 2,620 = 2,532 (microbes) + 88 (DNA resources)

(FY)

(644 strains for abroad)

antibiotic

Medical

Toxin Virulence factor Antibiotic resistance

Detection

NBRC Biological Resource Center, NITE

Food industry Bioremediation

Genetic recombination

biofuel

bioplastic

detergent

For microbial safety check

http://www.ac-illust.com/main/dl_info.php?id=27195&sw=%E7%B4%8D%E8%B1%86

1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013

Pyrococcus horikosii 1998.4.30

Aeropyrum pernix K1 1999.7.30

Staphylocuccus aureus N315 2001.4.20

Sulfolobus tokodaii strain 7 2001.9.13

Corynebacterium efficiens YS-314 2002.3.29

Staphylocuccus aureus MW2 2002.5.25

Staphylococcus heamolyticus JCSC1435 2005.6.30

Aspergillus oryzae RIB40 2005.12.21

Streptomyces avermitilis MA-4680 2003.4.8

Kocuria rhizophila DC2201 2008.3.31

Anaerolinea thermophila 2011.1.24 Acidiphilium multivorum 2011.3.29 Microlunatus phosphorus 2011.6.1 Sphingobium sp. SYK-6 2011.9.1 Saccharomyces cerevisiae K7 2011.9.9

Brevibacillus brevis 47 Rhodococcus erythropolis PR4 Rhodococcus opacus B4 Gemmatimonas aurantiaca T-27 2009.3.31 Desulfovibrio magneticus RS-1 2009.7.31 Acetobacter pasteurianus 2009.10.20

Kitasatospora setae 2010.11.11

Deferribacter desulfuricans 2010.3.5

Salmonella typhimurium 2010.11.26

Arthrospila platensis

Sphingobium japonicum 2010.3.29

Methanocella paludicola 2010.1.4

Tetragenococcus halophilus NBRC 12172 2013.6.18 Actinoplanes missouriensis NBRC 102363 2013.11.26

Caldisericum exile AZM16c01 Oscillibacter valericigenes Sjm18-20 2012.5.14 Arthrospira platensis NIES-39 2012.5.15

Streptomyces avermitilis MA-4680 2003.4.8

NITE Sequence capability / day

Analyzing and Supplying of genome information

NBRC strains

Genomics

ABI 3730XL (4) Roche GS 454 FLX (1)

Illumina HiSeq1000 (1) MiSeq (1)

DOGAN Genome sequence information

Gene cluster for secondary metabolite biosynthesis

DoBISCUIT

http://www.bio.nite.go.jp/dogan/top

http://www.bio.nite.go.jp/mifup/

31K cultures

Functional Potential database

MiFup

http://www.bio.nite.go.jp/pks/

Can I use microbe for foods?

(Bacterial) Pathogenic factor

Drug resistance ability Allergy factor

Newly developed database “MiFuP Safety” (Microbial Functional Potential search database for safety)

What is “MiFuP Safety”？

Estimate/detect functional genes that are pathogenic to humans and/or industrially harmful from the genomic information of microorganisms

NBRC analysis data

Public (literature) information

Draft genome or Partial sequences

Decrease the cost of genome analysis

gene a gene b B B B B

B

A Hazard

Gene set

C E D

gene c gene d gene e

Gene set

In order to make it possible to estimate the function from the

detected gene, a combination of genes necessary for exerting

harmful function is set based on the literature survey.

Detection condition of gene set

Hazard

Available to search 57 functions

Upload file or Copy & Paste the sequence to this window.

FASTA format (Available Multi-FASTA format）

選択

click

It was found that it has genes belonging to the thiol-activated cytolytic toxin family, a kind of membrane pore-forming toxin (toxin showing toxicity by puncturing the cell membrane).

Click here to see gene information page in detail

Display of the genes involved in the function (including partially hit)

Report Dpwnload

Function name

Link to the presumed sequence representation of the corresponding gene

Result

The genes constituting the toxin were partially detected. * It is presumed that it does not have the function as toxin because necessary genes are not available.

Detail information page

Mass (m/z)

A株

B株

C株

P1

Inte

nsit

y (%

)

11400 [email protected]

Adding the MALDI Matrix (consumable cost: approx. $0.20/sample)

Step2: Select biomarker candidates from the theoretical molecular weights of 53 types of ribosomal proteins

>Species A ATCGCATGCATGCATGCATGCATGCATGCATGCATGCATGC…. >Species B ATCGCATGCATGCATGCATGCATGCATGCATGCATGCATGC… >Species C ATCGCATGCATGCATGCATGCATGCATGCATGCATGCATGC… >Species D ATCGCATGCATGCATGCATGCATGCATGCATGCATGCATGC…

P1 P2 P3

specific proteins were selected

P1～n

Development of new method: Detection of specific

proteins (biomarkers) by MALDI-TOF MS Step1: Comparison of whole genome sequences

・・・・・・・・・・

Step3: MALDI-TOF MS data acquisition

Step4: Comparison between biomarker candidate and actual detected data

※Select biomarker candidates

※In this time we targeted ribosomal protein

Whole genome seq.

Detection of Bio-marker for microbial identification using MALDI-TOF MS

100 80 60 40 20 Similarity (%)

A. nosocomialis NBRC 110503 A. nosocomialis NBRC 110501 A. nosocomialis GTC 03313 A. nosocomialis NBRC 110499 A. nosocomialis NBRC 110498 A. nosocomialis LMG 10619T A. nosocomialis NBRC 110500

A. schindleri GTC 3703T A. lwoffii NBRC 109760T A. brisouii DSM 18516T A. tandoii KCTC 12417T A. johnsonii DSM 6963T A. bouvetii GTC 3697T A. pittii NBRC 110507 A. pittii NBRC 110509 A. pittii NBRC 110504 A. pittii NBRC 110506 A. pittii NBRC 110505 A. pittii NBRC 110508 A. pittii NBRC 110510 A. pittii DSM 25618T

A. oleivorans KCTC 23045T

A. calcoaceticus KCTC 2357T

A. nosocomialis NBRC 110502

A. nosocomialis GTC 03312 A. baumannii NBRC 110491 A. baumannii NBRC 109757T A. baumannii NBRC 110490 A. baumannii NBRC 110489 A. baumannii GTC 03328 A. baumannii GTC 03322 A. baumannii GTC 03323 A. baumannii NBRC 110492 A. baumannii GTC 03324 A. baumannii GTC 03325 A. baumannii GTC 03329 A. baumannii NBRC 110493 A. baumannii NBRC 110495 A. baumannii NBRC 110494

A. ursingii NBRC 110512 A. ursingii NBRC 110605 A. ursingii NBRC 110511 A. ursingii GTC 03702T

A. gerneri KCTC 12415T

A. bereziniae KCTC 23199T A. guillouiae KCTC 23200T A. haemolyticus NBRC 109758T A. parvus GTC 03704T A. venetianus GTC 14626T A. gyllenbergii GTC 14627T

Acinetobacter sp. NBRC 110496 A. beijerinckii GTC 14628T

A. tjernbergiae DSM 14971T A. junii NBRC 109759T A. junii NBRC 110497 A. baylyi GTC 3695T A. soli KCTC 22184T A. rudis DSM 24031T

A. indicus DSM 25388T A. towneri KCTC 12419T A. radioresistens NBRC 102413T

A. pittii

A. nosocomialis

A. baumannii

A. juniii

A. ursingii

Biomarker ensures reliable identification

A. lwoffii NBRC 109760T

A. brisouii DSM 18516T

A. pittii NBRC 110507



A. pittii NBRC 110506 A. pittii NBRC 110505



A. pittii DSM 25618T

A. oleivorans KCTC 23045T

A. calcoaceticus KCTC 2357T




A. nosocomialis GTC 03313

A. nosocomialis NBRC 110499 A. nosocomialis NBRC 110498

A. nosocomialis LMG 10619T


A. nosocomialis GTC 03312

A. baumannii NBRC 110491

A. baumannii NBRC 109757T



A. baumannii GTC 03328










A. baylyi GTC 3695T

A. indicus DSM 25388T

Microbial identification using MALDI-TOF MS biomarkers

Conclusion

• As a public Culture Collection, BRC serves as an infrastructure for collecting and providing biological resources and functional information (including genome information)

• More and more, microbial genome information will be used for various purposes. By effectively utilizing genome information, the microbial resource center can lead to better services of BRC.

Thank you for your attention.

nbrc genome sequencing projects for 20 years and current ......nbrc genome sequencing projects for...

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