NBRC genome sequencing projects for 20 years and current ......NBRC genome sequencing projects for...
Transcript of 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
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
Database Structure for gene search
sp|E0TVS5|XYLA_BACPZ
sp|P00944|XYLA_ECOLI
sp|Q9RFM4|XYLA_STRLI
1000
sp|P39305|ULAE_ECOLI
sp|B5F3B3|ULAE_SALA4
sp|P30147|HYI_ECOLI
1000
sp|Q8X840|FRLC_ECO57
sp|A7ZAH3|IOLI_BACA2
tr|B0M9Y4|B0M9Y4_9FIRM
tr|E5VM38|E5VM38_9FIRM
tr|D4MXK4|D4MXK4_9FIRM
tr|B0P019|B0P019_9CLOT
1000
tr|E5VY38|E5VY38_9FIRM
tr|B0MBA3|B0MBA3_9FIRM
1000
1000
tr|E5VLF2|E5VLF2_9FIRM
tr|D4MXC6|D4MXC6_9FIRM
tr|B0P2B9|B0P2B9_9CLOT
1000
tr|D1AP51|D1AP51_SEBTE
1000
980
820
tr|C1KKR1|C1KKR1_RHOSH
tr|L1K8E2|L1K8E2_9RHOB
tr|D0D8G1|D0D8G1_9RHOB
tr|Q0FST9|Q0FST9_9RHOB
1000
1000
tr|I8TMF0|I8TMF0_RHILT
tr|J2KE76|J2KE76_9RHIZ
tr|A6U5W8|A6U5W8_SINMW
728
999
1000
tr|J7LR16|J7LR16_9MICC
tr|A1R5X8|A1R5X8_ARTAT
tr|H0QL71|H0QL71_ARTGO
1000
tr|A8S4J7|A8S4J7_9CLOT
tr|G5HZV8|G5HZV8_9CLOT
tr|K2C6B1|K2C6B1_9BACT
1000
1000
494
tr|K1M201|K1M201_9BACT
tr|M7YC72|M7YC72_9BACT
tr|K0W6N1|K0W6N1_9BACT545
tr|A3HT29|A3HT29_9BACT
893
tr|G0J6J4|G0J6J4_CYCMS
407
tr|I3IAY6|I3 IAY6_9GAMM
1000
tr|C6W4S8|C6W4S8_DYAFD
tr|A3HUZ6|A3HUZ6_9BACT
775
1000
742
425
tr|F6FQM0|F6FQM0_ISOV2
tr|D7B7R0|D7B7R0_NOCDD
tr|F6FWM2|F6FWM2_ISOV2
673
tr|I1D005|I1D005_9PSEU
538
tr|D8JCN9|D8JCN9_HALJB
1000
719
986
1000
998
tr|B0MBJ8|B0MBJ8_9FIRM
tr|E5VR30|E5VR30_9FIRM
tr|B0PGH6|B0PGH6_9FIRM
tr|C0C310|C0C310_9CLOT
tr|J1H0W3|J1H0W3_9CLOT
tr|J0WYN5|J0WYN5_9CLOT
tr|D3AJP4|D3AJP4_9CLOT
750
1000
1000
tr|H1C8B9|H1C8B9_9FIRM
tr|G9YVF8|G9YVF8_9FIRM
tr|F7KP61|F7KP61_9FIRM
tr|B0NGC3|B0NGC3_EUBSP
1000
1000
sp|O50580|DT3E_PSECI
536
tr|F0RRH7|F0RRH7_SPHGB
tr|G8QSW3|G8QSW3_SPHPG
tr|G8QV10|G8QV10_SPHPG
994
852
1000
tr|B8I944|B8I944_CLOCE
tr|H2JAW2|H2JAW2_9CLOT
tr|F7KP59|F7KP59_9FIRM
tr|B0NGC5|B0NGC5_EUBSP
tr|C0C0P1|C0C0P1_9CLOT
1000
tr|N2ADA5|N2ADA5_9CLOT
1000
1000
548
tr|K5DVN1|K5DVN1_RHILU
tr|J0GUR9|J0GUR9_RHILV
tr|M8B3J5|M8B3J5_RHIRD
845
tr|G6XUT9|G6XUT9_RHIRD
tr|F5J831|F5J831_9RHIZ
tr|A9CH28|A9CH28_AGRT5
506
945
759
tr|N6UE14|N6UE14_9RHIZ
1000
517
1000
tr|H2J9K2|H2J9K2_9CLOT
744
tr|R0DE69|R0DE69_9CLOT
tr|A8RG82|A8RG82_9CLOT
tr|C6JHJ5|C6JHJ5_9FIRM
1000
513
1000
tr|F5SL39|F5SL39_9BACL
696
tr|B0N9L7|B0N9L7_EUBSP
284
tr|F3AXJ9|F3AXJ9_9FIRM
tr|A5KKM3|A5KKM3_9FIRM
tr|F7JD76|F7JD76_9FIRM
780
796
1000
tr|D4LXW6|D4LXW6_9FIRM
tr|N9ZCF1|N9ZCF1_9CLOT
tr|F0YVV0|F0YVV0_9CLOT
1000
827
688
281
148
684
330
1000
523
949
864
1000
203
605
750
328
372
258
874
586
sp|O05264|RHAA_BACSU
sp|P32170|RHAA_ECOLI
1000
0.05
Step1. Search for harmful genetic information
Step2. Identification of key genes ->> Determine the threshold value for detecting harmfulness
Step3. Estimate potential of hazard from genomic information
Investigate characteristic motifs common to the protein of interest
Investigation of threshold condition for homology search that can detect only target protein
Setting of conditions that can detect only proteins of the same origin as the target protein
Step2. detail
○Motif search ○Homology search ○Phylogenetic 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 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 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. 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. 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.