Roles and Quality Management of Culture Collection in Genomic

Roles and Quality Management of Culture Collections in Genomic Era

21 November 2018 WDCM GCM2.0 Workshop

127

Ken-ichiro Suzuki Department of Fermentation Science Faculty of Applied Bioscience Tokyo University of Agriculture (TUA) 鈴木健一朗東京農業大学応用生物科学部醸造科学科 Relationship between Culture Collection and Taxonomy (Summary of Today’s Talk)

Taxonomy

Scientific background

Providing Type strains

Academic Community

Social Infrastructure

Culture Collections and the Network

21/11/2018 Whole Genome Sequences – Summary of Today’s Talk –

• Research purpose – Application – Taxonomic comparison without transfer of strains – Taxonomy for In silico hybridization • Quality management of collection – Trace the transfer at strain level – Identity confirmation with the depositor – Identity confirmation with other culture collections 21/11/2018 Whole Genome Sequence Analysis for Taxonomic Studies

Courtesy of NBRC Culture Collection

21/11/2018 Relationship of 16s rRNA homology and DNA-DNA reassociation values

DNA-DNA reassociation (%) 0 10 20 30 40 50 60 70 80 90 100 100 ▽ × △△ △ ▲▽ ×△× × × ▼ ×△ △▽× △ ▽ ▽ × ▼ × ▽ 99 × ▼× × × ◎▽ 98 ◎◎ ◎ ××× ◎ ◎◎ 97 × ◎ ▼ ◎ ◎ ▼ ◎ 96 × ××× ×× ▼ 95 DNA threshold value

94 for species delineation 16S rRNA homology homology (%) rRNA 16S 93 × 92 × × × : Membrane filter method 91 △ ▲ ▽ ▼ : Renaturation method Stackebrandt & Goebel, 1994 ◎ : S1 nuclease method

21/11/2018 Principle of DNA-DNA Hybridization

One of the most essential methods to elucidate the species level Identification.

Fragmen- Heat tation denatured

Labeling Hybridization reaction (RI, fluorescent)

Organism A (Reference)

Heat Ratio = Index (ds) (ss) Immobilized Washing denatured

Organism B

21/11/2018 Judgement of the Results of DNA-DNA Hybridization (%)100 A: Species 90 B: Subspecies DNA A B C: Other subspecies

80 in the same species - DNA Relatedness 70 D: Other species C in different species 60 (J. L. Johnson 1973, 1984) 50 40 D The phylogenetic definition of

30 u species generally would include strains with 20 approximately 70% or greater 10 DNA-DNA relatedness and 0 with 5°C or less DTm.

21/11/2018 (Wayne, et al. 1987) Definition of Bacterial species Based on DDH

The species is the basic unit of bacterial taxonomy and is defined as a group of strains, including the type strain, sharing 70% or greater DNA-DNA relatedness with 5ºC or less ΔTm (Vandamme et al. 1996, Wayne et al. 1987).

However, DNA-DNA hybridization (DDH) is … There are several different methods. There are many parameters such as salt concentration, temperature, etc. It is easily affected by experimental error. It is required high-skilled techniques. There are times that the values may be different between the papers.

21/11/2018 In silico DNA-DNA Hybridization • Various methods have been developed for bacterial taxonomy using genome sequences. • The relationship between in silico DDH and traditional DDH was reported. • Advantage of In silico DDH is based on the absolute data of WGS

The average nucleotide identity (ANI) Konstantinidis & Tiedje, Proc Natl Acad Sci USA 2005, 102, 2567–2572 Goris et al. Int J Syst Evol Microbiol, 2007, 57, 81–91 Richter & Rosselló-Móra, Proc Natl Acad Sci USA, 2009, 106, 19126-19131. 95-96% ANI corresponds to 60-70% DDH.

Genome-To-Genome Distance Calculator (GGDC) Auch et al., Stand Gen Sci, 2010, 2, 117-134 Auch et al., Stand Gen Sci, 2010, 2, 142-148 0.0412 GGDC formula (2) value corresponds to 70% DDH.

DNA maximal unique matches index (MUMi) Deloger et al., J Bacterial 2009, 191, 91-99 0.33±0.03 MUMi corresponds to 95%±0.5% ANI.

21/11/2018 Case 1: Proposal of New Species Based on DDH

- Taxonomic Study of Novel Strains Belonging to the Family Demequinaceae Isolated Using SPPY Agar

Studied by Dr. Moriyuki Hamada, NBRC, Skerman Award Winner 2017

21/11/2018 NBRC Culture Collection 21/11/2018 Phylogenetic Tree of the Isolates Based on 16S rRNA Gene

Sequences 96 HI12-100 100 HI12-104 HI12-140 0.01 Knuc 61 HI12-128 100 HI12-44 HI12-143 71 HI12-121 85 Lysinimicrobium cluster 98 HI12-114 92 HI12-135 61 Lysinimicrobium mangrovi NBRC 105856T (AB639012) 91 HI12-122 HI12-123 61 HI12-45 HI12-111 63 Demequina flava NBRC 105854T (AB674956) 71 64 Demequina globuliformis NBRC 106266T (AB639018) 95 Demequina sediminicola NBRC 105855T (AB674957) 62 HI12-62 68 HI12-125 77 HI12-149 100 HI12-106 82 96 Demequina salsinemoris NBRC 105323T (AB639017) Demequina cluster HI12-66 100 HI12-147 89 HI12-71 Demequina aestuarii NBRC 106260T (AB639015) T 68 100 Demequina aurantiaca NBRC 106265 (AB639020) Demequina oxidasica NBRC 106264T (AB639019) 63 Demequina lutea NBRC 106155T (AB639016) Actinotalea fermentans NBRC 105374T (AB639014) 89 Cellulomonas flavigena DSM 20109T (X83799) 73 Cellulomonas cellasea DSM 20118T (X83804) 66 Cellulomonas bogoriensis 69B4T (X92152) Oerskovia turbata DSM 20577T (X83806) 100 Oerskovia enterophila DSM 43852T (X83807) Sanguibacter marinus 1-19T (AJ783958) 93 Sanguibacter keddieii ST-74T (X79450) 21/11/2018 Brevibacterium linens DSM 20425T (X77451) Polyphasic Approach by Chemotaxonomy

Characteristic 13 isolates Lysinimicrobium 7 isolates Demequina (Lysinimicrobium spp.) (Demequina spp.) Peptidoglycan type A4α A4α A4β A4β

Diamino acid L-Lys L-Lys L-Orn L-Orn

Interpeptide bridge D-Glu or D-Glu L-Ser–D-Glu or Ser–D-Asp or D-Ser–D-Glu D-Glu Ser–D-Glu

Major menaquinone DMK-9(H4) DMK-9(H4) DMK-9(H4) DMK-9(H4)

Major fatty acids ai-C15:0, ai-C17:0, ai-C15:0, ai-C17:0, ai-C15:0, ai-C17:0, ai-C15:0, ai-C17:0, C16:0 C16:0 C16:0 C16:0

Polar lipids§ DPG, PG, PI, PIMs, DPG, PG, PI, PIMs, DPG, PG, PI, DPG, PG, PI PL PL PIMs, PL

Genome size 2.68–3.23 Mbp 2.97Mbp 2.50–3.35 Mbp 2.46–3.21 Mbp

DNA G+C content 71.4–72.4 71.8 68.9–71.1 62.7–70.2

§DPG, Diphosphatidylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIMs, phosphatidylinositol-mannosides; PL, unknown polar lipid.

21/11/2018 ANIb Values and 16S rRNA Gene Sequence Similarities among the 13 Isolates and L. mangrovi NBRC 105856 Proposal of 9 New Species for 13 Isolates

16S rRNA Gene Sequence Similarity

ANIb

21/11/2018 Hamada et al. 2015 Comparison of the Dendrograms by 16S, MLSA and ANI

HI12-140 0.01 0.02 87 0.02 HI12-140 96 HI12-100 100 HI12-104 HI12-104 100 HI12-104 HI12-100 100 HI12-100 HI12-140 HI12-121 HI12-121 61 HI12-128 HI12-114 100 HI12-114 100 HI12-44 79 HI12-135 HI12-135 HI12-143 HI12-128 100 100 HI12-123 71 HI12-121 52 85 HI12-44 HI12-45 98 HI12-114 95 92 L. mangrovi HI12-135 HI12-122 61 HI12-143 L. mangrovi 100 HI12-44 91 HI12-123 HI12-122 HI12-128 99 100 HI12-45 HI12-123 HI12-143 HI12-122 61 HI12-45 L. mangrovi HI12-111 HI12-111 HI12-111 87 D. flava 63 D. flava D. salsinemoris 100 D. globuliformis 71 64 D. globuliformis 96 HI12-106 D. sediminicola 81 HI12-66 95 D. sediminicola 100 HI12-62 62 HI12-62 D. flava HI12-125 68 HI12-125 100 100 D. globuliformis 77 HI12-149 HI12-149 D. sediminicola HI12-147 HI12-106 100 HI12-62 66 HI12-71 82 96 D. salsinemoris HI12-125 83 D. aestuarii HI12-66 100 HI12-149 D. salsinemoris 100 HI12-147 100 HI12-71 89 HI12-71 HI12-106 100 HI12-147 D. aestuarii HI12-66 D. aestuarii 68 100 D. aurantiaca D. aurantiaca D. aurantiaca 100 D. oxidasica D. oxidasica D. oxidasica 63 D. lutea D. lutea D. lutea

16S rRNA gene MLSA ANI (Concatenated amino-acid sequences of atpD, gyrB, recA, rpoB and trpB genes) 21/11/2018 Conclusion

• 20 Strains which phylogenetically related to members of the family Demequinaceae were isolated from Iriomote Island, Japan, using the SPPY agar • Of them, 13 strains formed a monophyletic cluster with Lysinimicrobium mangrovi and remaining 7 strains were included in Demequina cluster • Major chemotaxonomic characteristics of the strains corresponded to those of the genera Lysinimicrobium and Demequina, respectively • Results of in silico DNA-DNA hybridization suggested that 20 strains should be assigned to 9 new species of the genus Lysinimicrobium and 7 new species of the genus Demequina, respectively

21/11/2018 Case 2. Speciation of the Genus Nocardia using Whole Genome Sequences

The genus Nocardia is a large genus of actinomycetes. The species of the genus are known not only as human/animal pathogen but also artificial organic Dr. Tomohiko compound decomposer and producers of various Tamura bioactive compounds. NBRC

Nocardia alba NBRC 108234 Nocardia caishijiensis NBRC 108228 Nocardia harenae NBRC 108248

21/11/2018 16S rRNA gene sequence ANI 98 Nocardia inohanensis IFM 0092 T (AB092560) Nocardia asteroides NBRC 15531 0.01 Nocardia yamanashiensis IFM 0265 T (AB092561) Nocardia salmonicida NBRC 13393 Nocardia niigatensis DSM 44670 T (DQ659910) Nocardia soli NBRC 100376 Nocardia cummidelens NBRC 100378 Nocardia concava IFM 0354 T (AB126880) Nocardia alba NBRC 108234 Nocardia uniformis DSM 43136 T (AF430044) Nocardia ignorata NBRC 108230 Nocardia otitidiscaviarum DSM 43242 T (AF430067) Nocardia coubleae NBRC 108252 Nocardia crassostreae JCM 10500 T (AF430049) Nocardia caishijiensis NBRC 108228 Nocardia nova JCM 6044 T (AF430028) Nocardia thailandica NBRC 100428 Nocardia pseudobrasiliensis DSM 44290 T (AF430042) Nocardia harenae NBRC 108248 Nocardia miyunensis 117 T (AY639901) Nocardia cyriacigeorgica NBRC 100375 84 Nocardia jiangxiensis 43401 T (AY639902) Nocardia jinanensis NBRC 108249 Nocardia terpenica IFM 0706 T (AB201298) Nocardia testacea NBRC 100365 Nocardia mexicana DSM 44952 T (FR733723) Nocardia grenadensis NBRC 108939 Nocardia anaemiae IFM 0323 T (AB162801) Nocardia sienata NBRC 100364 Nocardia pseudovaccinii DSM 43406 T (AF430046) Nocardia rhamnosiphila NBRC 108938 Nocardia vinacea MK703-102F1 T (AB024312) "Nocardia fusca" NBRC 14340 Nocardia speluncae NBRC 108251 Nocardia caishijiensis F829 T (AF459443) Nocardia flavorosea NBRC 108225 Nocardia alba YIM 30243 T (AY222321) Nocardia carnea NBRC 14403 Nocardia jejuensis N3-2 T (AY964666) Nocardia paucivorans NBRC 100373 99 Nocardia ignorata DSM 44496 T (AJ303008) Nocardia brevicatena NBRC 12119 Nocardia coubleae OFNN11 T (DQ235688) Nocardia sp. NBRC 108935 99 Nocardia salmonicida DSM 40472 T (AF430050) Nocardia takedensis NBRC 100417 96 Nocardia soli DSM 44488 T (AF430051) Nocardia puris NBRC 108233 99 Nocardia cummidelens DSM 44490 T (AF430052) Nocardia amikacinitolerans NBRC 108937 99 Nocardia gamkensis CZH20 T (DQ235272) Nocardia xishanensis NBRC 101358 Nocardia exalbida IFM 0803 T (AB187522) Nocardia lijiangensis NBRC 108240 Nocardia lijiangensis YIM 33378 T (AY779043) 0.02 Nocardia shimofusensis NBRC 100134 Nocardia xishanensis AS4.1860 T (AY333115) Nocardia higoensis NBRC 100133 Nocardia farcinica NBRC 15532 99 Nocardia brevicatena DSM 43024 T (AF430040) Nocardia vinacea NBRC 16497 Nocardia paucivorans DSM 44386 T (AF430041) Nocardia pseudovaccinii NBRC 100343 Nocardia asteroides DSM 43757 T (AF430019) Nocardia anaemiae NBRC 100462 Nocardia thailandica IFM 10145 T (AB126874) Nocardia sp. NBRC 108936 Nocardia abscessus IMMIBD-1592 T (AF218292) Nocardia brasiliensis NBRC 14402 Nocardia cyriacigeorgica DSM 44484 T (AF430027) Nocardia tenerifensis NBRC 101015 87 Nocardia jinanensis 04-5195 T (DQ462650) Nocardia altamirensis NBRC 108246 99 Nocardia speluncae N2-11 T (AM422449) Nocardia pneumoniae NBRC 100136 Nocardia grenadensis GW5-5797 T (FR729900) Nocardia amamiensis NBRC 102102 78 Nocardia pigrifrangens 7031 T (AF219974) Nocardia niwae NBRC 108934 Nocardia carnea DSM 43397 T (AF430035) Nocardia gamkensis NBRC 108242 97 99 Nocardia testacea JCM 12235 T (AB192415) Nocardia exalbida NBRC 100660 Nocardia sienata IFM 10088 T (AB121770) Nocardia arthritidis NBRC 100137 Nocardia beijingensis NBRC 16342 Nocardia rhamnosiphila 202GMO T (EF418604) Nocardia araoensis NBRC 100135 Nocardia flavorosea JCM 3332 T (Z46754) Nocardia asiatica NBRC 100129 Nocardia transvalensis DSM 43405 T (AF430047) Nocardia abscessus NBRC 100374 Nocardia harenae WS-26 T (DQ282122) Nocardia uniformis NBRC 13702 Nocardia takedensis MS1-3 T (AB158277) Nocardia otitidiscaviarum NBRC 14405 Nocardia tenerifensis (AJ556157) Nocardia yamanashiensis NBRC 100130 78 Nocardia brasiliensis DSM 43758 T (AF430038) Nocardia inohanensis NBRC 100128 Nocardia altamirensis DSM 44997 T (EU006090) Nocardia niigatensis NBRC 100131 Nocardia vaccinii DSM 43285 T (AF430045) Nocardia concava NBRC 100430 Nocardia vermiculata IFM 0391 T (AB126873) Nocardia jejuensis NBRC 103114 Nocardia elegans IMMIBN-402 T (AJ854057) Nocardia crassostreae NBRC 100342 74 Nocardia mikamii W8061DSM 45174 T (EU484388) Nocardia acidivorans NBRC 108247 Nocardia cerradoensis Y9 T (AF060790) Nocardia terpenica NBRC 100888 73 Nocardia pseudobrasiliensis NBRC 108224 Nocardia aobensis IFM 0372 T (AB126876) Nocardia transvalensis NBRC 15921 Nocardia africana DSM 44491 T (AF430054) Nocardia mexicana NBRC 108244 Nocardia veterana DSM 44445 T (AF430055) Nocardia vaccinii NBRC 15922 97 Nocardia kruczakiae MB2876ATCC BAA-948 T (AY441974) Nocardia miyunensis NBRC 108239 88 Nocardia pneumoniae IFM 0784 T (AB108780) Nocardia jiangxiensis NBRC 101359 Nocardia amamiensis TT00-78 T (AB275164) Nocardia vermiculata NBRC 100427 80 Nocardia niwae W9241CCUG 57756 T (FJ765056) Nocardia veterana NBRC 100344 Nocardia beijingensis AS4.1521T T (AF154129) Nocardia cerradoensis NBRC 101014 Nocardia araoensis IFM 0575 T (AB108779) Nocardia mikamii NBRC 108933 Nocardia arthritidis IFM 10035 T (AB108781) Nocardia kruczakiae NBRC 101016 Nocardia puris IMMIBR-145 DSM 44599 T (AJ508748) "Nocardia violaceofusca" NBRC 14427 Nocardia asiatica IFM 0245 T (AB092566) Nocardia aobensis NBRC 100429 Nocardia nova NBRC 15556 Nocardia farcinica M.GoodfellowN898/ATCC 3318 T (Z36936) Nocardia elegans NBRC 108235 21/11/2018Nocardia shimofusensis YZ-96 T (AB108775) Nocardia africana NBRC 100379 Nocardia higoensis IFM 10084 T (AB108778) Rhodococcus erythropolis PR4 Conclusion • Draft sequences of type strains of 70 valid Nocardia species were analyzed. • In silico DDH by mean of ANI gave results that agrees with the taxonomic relationships based on MLSA and Bidirectional Best Hit (BBH) in the genera Nocardia • 98.9 -99.0 % BBH corresponds to 96-97 % ANI and 98.6- 98.8 % similarity of concatenated sequence (atpD-groL1- groL2-recA-rpoA-secY-sodA-ychY) corresponds to 96-97 % ANI in the genus Nocardia. • Possible synonymies of some species were shown from in silico DDH. • In silico DDH, BBH and MLSA will be useful tool for actinomycete taxonomy by accumulating data 21/11/2018 Quality Management of Culture Collections

in Genomic Era

NBRC Culture Collection

21/11/2018 Mission of Culture Collection

• Formerly, the task of a culture collection was to preserve microbial strains as they were deposited with the name that the depositor identified. • Now the quality requested by the users is dependent on the purpose of use. Culture collections are always responsible for the correct identification and updated names.

21/11/2018 Essential Functions of Culture Collections Collection, Preservation and Supply of: 1. Reference organisms for quality control and standardized tests  Needs of specification of strains by national or international regulations 2. Taxonomic type strains of prokaryotes (bacteria, archaea) and yeasts  According to the Bacteriological Code of Nomenclature  Living culture is also recommended for fungi derived from the type according to the Botanical Code 3. Materials for research and development  Characteristic for BRC and the original country for screening sources  Strains for large scale screening

21/11/2018 2. Quality Management of Culture Collections

1. Viability and stability Growth on appropriate media Revival from the preserved stock Estimation of long-term preservation 2. Purity Colony appearance, cell morphology Molecular technique (fingerprint, primer amplification) 3. Identity (Depositor>Collection, In-house maintenance) 16S rRNA gene sequences for prokaryotes Molecular technique (functional genes) MALDI-TOM/MS

21/11/2018 Case 1. Identity of P. subterranea SL1T & P. lettingae TMOT Revealed by Whole Genome Sequence Analysis

Pseudothermotoga subterranea (Jeanthon et al. 2000) Bhandari and Gupta 2014 NBRC 107924T <- DSM 9912T <- C. Jeanthon, SL1T

Pseudothermotoga lettingae (Balk et al. 2002) Bhandari and Gupta 2014 NBRC 107922T <- DSM 14385T <- M. Balk, TMOT (Other collcetion No.: ATCC BAA-301T)

Studied by Dr. Koji Mori, NBRC 21/11/2018 Genomic Identity of Pseudothermotoga subterranea SL1T & Pseudothermotoga lettingae TMOT

Thermotoga subterranea Jeanthon et al. 2000 C. Jeanthon et al. Thermotoga subterranea sp. nov., a new thermophilic bacterium isolated from a continental oil reservoir. Arch. Microbiol. (1995) 164: 91-97 Thermotoga lettingae Balk et al. 2002 M. Balk et al. Thermotoga lettingae sp. nov., a novel thermophilic, methanol-degrading bacterium isolated from a thermophilic anaerobic reactor. Int. J. Syst. Evol. Microbiol. (2002) 52: 1361-1368.

Pseudothermotoga subterranea (Jeanthon et al. 2000) Bhandari and Gupta 2014 Pseudothermotoga lettingae (Balk et al. 2002) Bhandari and Gupta 2014 V. Bhandari & R. S. Gupta. Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations. Antonie van Leeuwenhoek (2014) 105: 143-168

21/11/2018 Whole genome of P. subterranea SL1T & P. lettingae TMOT

P. lettingae DSM 14385T O. Zhaxybayeva et al. On the chimeric nature, thermophilic origin, and phylogenetic placement of the Thermotogales. Proc. Natl. Acad. Sci. USA (2009) 106: 5865-5870.

P. subterranea NBRC 107924T K. Mori et al. Thermotoga profunda sp. nov., and Thermotoga caldifontis sp. nov., anaerobic thermophilic bacteria isolated from terrestrial hot springs. Int. J. Syst. Evol. Microbiol. (2014) 64: 2128-2136. (In this study, we determined the whole genome sequences of 5 strains of Thermotoga (Pseudothermotoga) species including P. subterranea NBRC 107924T but practically we did not describe the result of . subterranea NBRC 107924T.)

21/11/2018 Relationship Based on 16S rRNA and DDH (ANI)

Phylogenetic tree based on 16S rRNA gene sequences

16S rRNA ANI

T.profunda P.elfii P.subterranea P.lettingae T.caldifontis T.hypogea P.thermarum T.maritima T. profunda 96.0 96.0 96.0 94.9 94.6 93.7 90.2 P. elfii 70.8 100.0 100.0 94.7 94.4 93.3 91.6 P. subterranea 70.5 99.1 100.0 94.7 94.4 93.3 91.6 P. lettingae 70.5 99.2 100.0 94.7 94.4 93.3 91.6 T. caldifontis 65.9 65.2 65.1 65.3 98.6 96.2 92.5 T. hypogea 66.1 65.4 65.3 65.5 76.6 95.7 92.5 P. thermarum 67.2 66.6 66.5 66.6 65.7 66.0 93.0 T. maritima 64.2 64.6 64.9 65.2 66.1 65.6 64.7

21/11/2018 CONCLUSION We concluded that P. subterranea NBRC 107924T and P. lettingae NBRC 107922T (=DSM 14385T) are the same strain.

The reason is that the whole genome sequence of P. subterranea NBRC 107924T is completely identical with that of P. lettingae DSM 14385T.

Communication and custom of exchange among culture collection contributed to this solution.

Different from wet DDH, WGS-based DDH provides absolute data accompanying with the strain of CC.

21/11/2018 Case 2: Response of Culture Collection to Reclassification

• Proposal of a new subspecies

21/11/2018 Proposal of a New Subspecies

21/11/2018 Differentiation Based on DNA-DNA Relatedness

21/11/2018 Description of the New Subspecies

21/11/2018 Reclassification by a Culture Collection

Publication (Taxonomic study) Species A (strain 1T and strain 2)  Species A (strain 1T) + Species B (strain 2T)

In response to this publication, culture collection must change the catalogue database (Strain-based database) as follows: Before After Species A strain 1T Species A strain 1T strain 2 Species B strain 2T strain 3 ? strain 3 strain 4 ? strain 4 Needs reidentification by CC 21/11/2018 21/11/2018 On-Line Catalogue of NBRC

16S rDNA

21/11/2018 Determination of 16S rRNA gene Sequences by Culture Collections for Quality Management

• Necessary condition to confirm the identity of the deposited strain to that the depositor studied. • Not sufficient condition but the sequence data is a core of the proposal of a new species • The possibility of new taxa can be estimated by the data of 16S rRNA sequences.

21/11/2018 Re-identification of Strains in a Culture Collection (Updating of the Names)

• Many of old NBRC strains including former IFO strains that have been deposited since 1950s were bearing names identified only phenotypically. • Classification has been drastically improved since 1980s, when molecular methods were introduced into the taxonomy.

We have to verify whether their scientific names are still correct now.

• NBRC is trying to determine 16S rRNA gene sequences (prokaryotes) and ITS and D1/D2 regions of 26S rRNA (eukaryotes) for all open NBRC strains (more than 15,000) deposited from 1950s.

• These sequence data are publicized and available at NBRC on-line catalogue.

21/11/2018 QC Flow from Accession to Issuing Certificate Depositor

First contact Material (Culture) Reason of deposit Additional data •Notice of Data sheet containing (Special check points) acceptance Taxon (possible name) Signed Accession •Accession Taxonomic information form number 16S rRNA gene •Certificate of sequence availability Source / location Check (material level) •(Depositor’s Terms & condition Preliminary check check) BSL (viability, purity etc.) 16S rRNA gene Check (Document level) Y / N sequence matching Culture Collection

21/11/2018 Proposal of a New Species of the Prokaryotes (Published in IJSEM)

AuthorCan GCM2.0 support this Manuscript process? Revised Containing Manuscript Proposing name with •Review comments Description (Etymology WGS Added and protologue) of taxon •Request of Time and 16S rRNA gene sequence WGS Cost for Assignment of type WGS strain with CC Certificate (WGS)

Check (Document level) Accepted! IJSEM Editor and Editorial Office

21/11/2018 Contribution of GCM2.0 Expected

1. If WGS is mandatory for proposal of a new species, all the published new species have WGS at publication. 2. Support of GCM2.0 in WGS determination for proposal of a new species before submission will be more valuable. 3. For that system, WDCM must evaluate the new species candidates before submission. 4. 16S rRNA gene sequence data are important especially confirmed by culture collections.

21/11/2018 Quality Management of JCM Accept of Deposit (Material Level)

Result Judgement Total Non- Year Contami Withdraw Deposit Dead identity OK NG NG Ratio nation al of 16S 2010 870 4 3 9 1 853 17 2.0%

2011 999 17 11 22 2 947 52 5.2% 2012 471 15 7 17 3 429 42 8.9% 2013 744 29 13 41 5 656 88 11.8% 2014 1035 56 28 60 5 885 150 14.5%

Data: Courtesy of Dr. T. Kudo, JCM These data indicate how important the quality management of culture collections for the quality of taxonomy. 21/11/2018 Advantage of WGS for Culture Collections • The WGS information contributes to the quality management of culture collections • The WGS information is valuable for the clients for their use of microbial resources • The WGS information will contribute to the sales of cultures of culture collections. • It may be worth for culture collections to collaborate with GCM2.0

21/11/2018 Second Stage of WGS for Culture Collections • The WGS information will indicate the genome-based biodiversity of microbial resources. • All microbiologists will recognize the usefulness of mirobial resources in culture collections • Correct identification of the non-type strains

21/11/2018 Thank you very much for your attention.

21/11/2018 News : Genome sequencing data required with Taxonomic Descriptions.

• IJSEM will soon be asking authors to provide genome sequencing data with descriptions of novel taxa in Taxonomic Descriptions. • Although not mandatory for publication, the inclusion of this data is highly recommended and will be expected to be included. If authors are unable to provide genome sequencing data for any reason this should be stated as such in their covering letter; exemptions will be considered on a case by case basis by the handling Editor. • Genome sequences are of great value to the systematics of prokaryotes. In addition to improving the general understanding of the biology of microorganisms, they improve the identification of prokaryotic species, identification of functional characteristics useful for resolving taxonomic groups and the resolution of the phylogeny of higher taxa. • This requirement will come into effect from January, 2018 and we ask that authors deposit the data in an established, freely available public database that does not require viewer registration (i.e. GenBank, ENA or DDBJ). • For any queries regarding these requirements, please email.

21/11/2018 Website of IJSEM Critical Issues of Use of Microorganisms from Culture Collection 1. Correct identification 2. Quality of materials satisfying purpose of use 3. Provision of sufficient information with materials 4. Terms and conditions of use especially for commercialization 5. Compliance with laws and regulations for transfer and use of materials

21/11/2018 SUMMARY for Whole Genome Analysis

• Whole genome analysis may find closely related species (type strain) for reclassification especially for the synonymy in such big genus without wet DNA-DNA hybridization. • Whole genome analysis has made it possible to reveal the potential of strains for use as well as the taxonomic analysis. • Whole genome sequences are essential data to develop differentiation with high resolution.

21/11/2018