Open Access

Non-contiguous finished genome sequence of Prevotella timonensis type strain 4401737T

Standards in Genomic Sciences20149:9031344

DOI: 10.4056/sigs.5098948

Published: 15 June 2014

Abstract

Prevotella timonensis strain 4401737T is a member of the genus Prevotella, which contains anaerobic Gram-negative bacteria. It was isolated from a human breast abscess. In this work, we describe a set of features of this organism, together with the complete genome sequence and annotation. The 3,169,464 bp long genome contains 2,746 protein-coding genes and 56 RNA genes, including 3 or 4 rRNA operons.

Keywords

Prevotella timonensis Bacteroidetes

Introduction

Prevotella timonenis strain 4401737T(CIP 108522T= CCUG 50105T) is the type strain of P. timonensis. This bacterium was isolated from a human breast abscess [1]. The genus Prevotella is comprised of anaerobic Gram-negative bacteria. It currently contains 47 members [2]. Recently, many species of the genus Prevotella have been isolated from human sources, often associated with the oral cavity [38], but also from feces [9], amniotic fluid [10], blood cultures, lung abscess pus, broncho-alveolar lavages [11] and pleural fluids [12].

Here we present a summary classification and a set of features for P. timonensis, together with the description of the non-contiguous finished genomic sequencing and annotation.

Classification and features

The 16S rRNA gene sequence of P. timonensis strain 4401737T was compared with sequences deposited in the Genbank database, indicating that the initial taxonomic classification is correct.

Figure 1 shows the phylogenetic neighborhood of P. timonensis in a 16S rRNA based tree.
Figure 1.

Part of a phylogenetic tree highlighting the position of Prevotella timonensis strain 4401737T relative to other type strains within the genus Prevotella by comparison of 16S rRNA gene sequences. GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALX, and phylogenetic inferences obtained using the neighbor joining method within the MEGA 5 software [13]. Numbers at the nodes are percentages of bootstrap values (≥ 50%) obtained by repeating the analysis 1,000 times to generate a majority consensus tree. Paraprevotella clara was used as the outgroup (not shown). The scale bar represents 0.002 nucleotide change per nucleotide position.

The bacterium was first characterized in 2004; it was isolated from a 40-year-old woman who underwent a breast abscess puncture. The organism was in the liquid from the punctured abscess and was cultured in the Timone Hospital microbiology laboratory.

Cells are rods 0.8–1.4 µm long and 0.3–0.5 µm wide and usually occurred singly. Optimal growth of strain 4401737T occurs at 37°C with a range for growth between 25 and 37 °C. After 72 hours growth on blood sheep agar at 37°C, surface colonies are circular, white-greyish, smooth, shiny, non-pigmented and 1–2 mm in diameter. Carbon sources utilized include ribose, glucose, lactose, maltose and tagatose. Activities of alkaline phosphatase, β-galactosidase, α-glucosidase, N-acetyl-β-glucosaminidase, α fucosidase, arginine arylamidase, leucyl glycine arylamidase, alanine arylamidase are detected. The fatty acid profile is characterized by the predominance of C14:0 (19.5%), C16:0 (15.3%), iso-C14:0 (14%) and a mixture of C18:2 ω6,9c and C18:0 (16%). The size and ultrastructure of cells were determined by negative staining transmission electron microscopy. The rods were 0.8–1.4 µm long and 0.3–0.5 µm wide (Figure 2, Table 1).
Figure 2.

Transmission electron micrograph of T. timonensis strain 4401737T, using a Morgani 268D (Philips) at an operating voltage of 60kV. The scale bar represents 500 µm.

Table 1.

Classification and general features of Prevotella timonensis strain 4401737T

MIGS ID

Property

Term

Evidence codea

 

Current classification

Domain Bacteria

TAS [14]

 

Phylum Bacteroidetes

TAS [15,16]

 

Class Bacteroidia

TAS [15,17]

 

Order Bacteroidales

TAS [15,18]

 

Family Prevotellaceae

TAS [15,19]

 

Genus Prevotella

TAS [2022]

 

Species Prevotella timonensis

TAS [1]

 

Type strain 4401737T

TAS [1]

 

Gram stain

Negative

TAS [1]

 

Cell shape

Rod-shaped

TAS [1]

 

Motility

Non motile

TAS [1]

 

Sporulation

Non-sporulating

TAS [1]

 

Temperature range

Mesophile

TAS [1]

 

Optimum temperature

37°C

TAS [1]

MIGS-6.3

Salinity

Not reported

 

MIGS-22

Oxygen requirement

Anaerobic

TAS [1]

 

Carbon source

Glucose, lactose, maltose, ribose, tagatose

TAS [1]

 

Energy source

Chemoorganotroph

NAS

MIGS-6

Habitat

Host

TAS [1]

MIGS-15

Biotic relationship

Free living

TAS [1]

MIGS-14

Pathogenicity

Unknown

NAS

 

Biosafety level

2

 
 

Isolation

Human breast abscess

 

MIGS-4

Geographic location

Marseille, France

TAS [1]

MIGS-5

Sample collection time

2004

TAS [1]

MIGS-4.1

Latitude

43°18 N

IDA

MIGS-4.1

Longitude

5°23 E

IDA

MIGS-4.3

Depth

Surface

IDA

MIGS-4.4

Altitude

21 m above sea level

IDA

Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [23]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

Genome sequencing and annotation

Genome project history

The organism was selected for sequencing on the basis of its phylogenetic position and 16S rDNA similarity to other members of the genus Prevotella, and is part of study of the new species characterized in our laboratory. A summary of the project information is shown in Table 2. The EMBL accession number is CBQQ010000001 and consists of 148 contigs (≥ 500 bp) and 25 scaffolds (> 1,500 bp). Table 2 shows the project information and its compliance with MIGS version 2.0 standards.
Table 2.

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

High-quality draft

MIGS-28

Libraries used

One paired end 3-kb library and two Shotgun libraries

MIGS-29

Sequencing platforms

454 GS FLX Titanium

MIGS-31.2

Fold coverage

78.12×

MIGS-30

Assemblers

Newbler version 2.5.3

MIGS-32

Gene calling method

Prodigal

 

EMBL ID

CBQQ010000001

 

EMBL Date of Release

June 18, 2013

 

Project relevance

Study of new species isolated in the URMITE

Growth conditions and DNA isolation

P. timonensis strain 4401737T was grown anaerobically on 5% sheep blood-enriched Columbia agar at 37°C. Five petri dishes were spread and colonies resuspended in 3 ml of TE buffer. Three hundred µl of 10% SDS and 150 µl of proteinase K were then added and incubation was performed over-night at 56°C. The DNA was then extracted using the phenol/chloroform method. The yield and the concentration were measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 84.3 ng/µl.

Genome sequencing and assembly

Shotgun and 3-kb paired-end sequencing strategies were performed. A shotgun library was constructed with 500 ng of DNA with the GS Rapid library Prep kit (Roche). For the paired-end sequencing, 5 µg of DNA was mechanically fragmented on a Hydroshear device (Digilab) with an enrichment size at 3–4 kb. The DNA fragmentation was visualized using the 2100 BioAnalyzer (Agilent) on a DNA labchip 7500 with an optimal size of 3.7 kb. The library was constructed according to the 454 GS FLX Titanium paired-end protocol. Circularization and nebulization were performed and generated a pattern with an optimal size of 574 bp. After PCR amplification through 17 cycles followed by double size selection, the single stranded paired-end library was then quantified using the Genios fluorometer (Tecan) at 1070 pg/µL. The library concentration equivalence was calculated as 3.42 × 109 molecules/µL. The library was stored at −20°C until further use. Another shotgun library was constructed with 1µg of DNA as described in the Rapid Library Preparation Method Manual GS FLX+ Series – XL+ except that fragmentation was obtained on Covaris® M220 focused-ultrasonocatorTM instead of on a Hydroshear device.

The shotgun and paired-end libraries obtained with the GS-FLX Titanium technology were clonally-amplified with 1 cpb in 4 SV-emPCR reactions, and 0.5 cpb in 2 SV-emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were 18.7% and 10.9%, respectively, in the 5 to 20% range from the Roche procedure. The shotgun library obtained with the GS-FLX+ technology was clonally-amplified with 3 cpb in 2 SV-emPCR reactions. The yield of the emPCR was 23.95%. Approximately 790,000 beads for the shotgun application and for the 3kb paired end were loaded on the GS Titanium PicoTiterPlate PTP Kit 70x75 and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche). The run was performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 573,130 passed filter wells were obtained and generated 249.97 Mb with an average length of 424 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 25 scaffolds and 105 large contigs (>1,500 bp).

Genome annotation

Open Reading Frames (ORFs) were predicted using Prodigal [24] with default parameters but the predicted ORFs were excluded if they were spanning a sequencing GAP region. The predicted bacterial protein sequences were searched against the GenBank database [25] and the Clusters of Orthologous Groups (COG) databases [26] using BLASTP. The tRNAscan-SE tool [27] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [28]. Transmembrane domains and signal peptides were predicted using TMHMM [29] and SignalP [30], respectively. ORFans were identified if their BLASTp E-value was lower than 1 × 10−3 for alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1 × 10−5. Such parameter thresholds have been used in previous works to define ORFans.

To estimate the mean level of nucleotide sequence similarity at the genome level between P. timonensis and Prevotella genomes available to date, we compared the only those ORFs only that could be found on the RAST server [31] with a query coverage of ≥60% and a minimum nucleotide length of 100 bp.

Genome properties

The genome is 3,169,464 bp long with a 40.50% GC content (Table 3, Figure 3). Of the 2,802 predicted genes, 2,746 were protein-coding genes, and 56 were RNAs. A total of 1,795 genes (65.37%) were assigned a putative function. 198 genes were identified as ORFans (7,21%). The remaining genes were annotated as hypothetical proteins (673 genes (24,51%)). The remaining genes were annotated as either hypothetical proteins or proteins of unknown function. The distribution of genes into COGs functional categories is presented in Table 4. The properties and the statistics of the genome are summarized in Tables 3 and 4.
Figure 5.

Graphical circular map of Prevotella timonensis genome. From outside to the center: Contigs (red / grey), COG category of genes on the forward strand (three circles), genes on forward strand (blue circle), genes on the reverse strand (red circle), COG category on the reverse strand (three circles), GC content.

Table 3.

Nucleotide content and gene count levels of the genome

Attribute

Value

% of totala

Genome size (bp)

3,169,464

100

DNA coding region (bp)

2,758,009

87.02

DNA G+C content (bp)

1,347,151

42.50

Total genes

2,802

100

RNA genes

56

2.00

Protein-coding genes

2,746

98.00

Genes with function prediction

1,795

65.37

Genes assigned to COGs

1,479

53.86

Genes with peptide signals

678

24.69

Genes with transmembrane helices

540

19.66

a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.

Table 4.

Number of genes associated with the 25 general COG functional categories

Code

Value

% of total

Description

J

135

4.92

Translation

A

0

0

RNA processing and modification

K

87

3.17

Transcription

L

179

6.52

Replication, recombination and repair

B

0

0

Chromatin structure and dynamics

D

23

0.84

Cell cycle control, mitosis and meiosis

Y

0

0

Nuclear structure

V

44

1.60

Defense mechanisms

T

42

1.53

Signal transduction mechanisms

M

171

6.23

Cell wall/membrane biogenesis

N

2

0.07

Cell motility

Z

0

0

Cytoskeleton

W

0

0

Extracellular structures

U

35

1.27

Intracellular trafficking and secretion

O

73

2.66

Posttranslational modification, protein turnover, chaperones

C

74

2.69

Energy production and conversion

G

107

3.90

Carbohydrate transport and metabolism

E

87

3.17

Amino acid transport and metabolism

F

62

2.26

Nucleotide transport and metabolism

H

62

2.22

Coenzyme transport and metabolism

I

42

1.53

Lipid transport and metabolism

P

98

3.57

Inorganic ion transport and metabolism

Q

12

0.44

Secondary metabolites biosynthesis, transport and catabolism

R

206

7.5

General function prediction only

S

80

2.91

Function unknown

X

1267

46.14

Not in COGs

The total is based on the total number of protein coding genes in the annotated genome.

Comparison with other Prevotella genomes

To date 33 genomes from species belonging to the genus Prevotella have been sequenced.

Whole genome sizes ranged between 2.42 Mb (P. bivia and P. amnii) and 3.62 Mb (P. ruminicola). The G+C content of the genomes was was between 36.5% for P. amnii and 55.9% for P. dentalis. 16S rRNA gene sequence comparison was performed to obtain phylogenetic analysis of Prevotella species. A cluster including P. bergensis, P. dentalis, P. multisaccharivorax, P. buccae, P. baroniae, P. dentasini, P. denticola and P. multiformis was identified. From this group. the genomes of P. bergensis, P. dentalis, P. multisaccharivorax, P. buccae, P. denticola and P. multiformis have been sequenced. It is interesting to note that these genomes showed the highest G+C contents (47.6-55.9%) among the bacteria included in the genus Prevotella. A more in-depth study will allow us to determine if this group of bacteria represent a particular evolutionary lineage.

The genome of another strain of the species P. timonensis was sequenced, strain CRIS 5C B1. The genome of P. buccalis, which is the more closely related species to P. timonensis when 16S rRNA encoding gene sequences were compared, has also been sequenced.P. timonensis strain 4401737T shared a mean sequence similarity of 96.45% (60.2-100%) with P. timonensis strain CRIS 5C B1 and of 84.02% (60–100%) with P. buccalis.

The partition of the coding sequences into subsystems [31] is similar for the two genomes except for the transposable elements, whose numbers are significantly higher in strain 4401737T.

Declarations

Acknowledgements

The authors thank Mr. Julien Paganini at Xegen Company (www.xegen.fr) for automating the genomic annotation process and Laetitia Pizzo, Audrey Borg and Audrey Averna for their technical assistance.

Authors’ Affiliations

(1)
Aix Marseille Université, URMITE, Faculté de médecine, Aix-Marseille Université

References

  1. Glazunova OO, Launay T, Raoult D, Roux V. Prevotella timonensis sp. nov., isolated from a human breast abscess. Int J Syst Evol Microbiol 2007; 57:883–886. PubMed http://dx.doi.org/10.1099/ijs.0.64609-0View ArticlePubMedGoogle Scholar
  2. Euzéby JP. List of Bacterial Names with Standing in Nomenclature: a folder available on the Internet. Int J Syst Bacteriol 1997; 47:590–592. PubMed http://dx.doi.org/10.1099/00207713-47-2-590View ArticlePubMedGoogle Scholar
  3. Downes J, Wade WG. Prevotella fusca sp. nov. and Prevotella scopos sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2011; 61:854–858. PubMed 1350 http://dx.doi.org/10.1099/ijs.0.023861-0View ArticlePubMedGoogle Scholar
  4. Downes J, Tanner ACR, Floyd E, Dewhirst FE, Wade WG. Prevotella saccharolytica sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2010; 60:2458–2461. PubMed http://dx.doi.org/10.1099/ijs.0.014720-0PubMed CentralView ArticlePubMedGoogle Scholar
  5. Sakamoto M, Natsuko Suzuki N, Okamoto M. Prevotella aurantiaca sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2010; 60:500–503. PubMed http://dx.doi.org/10.1099/ijs.0.012831-0View ArticlePubMedGoogle Scholar
  6. Downes J, Hooper SJ, Melanie J, Wilson MJ, Wade WC. Prevotella histicola sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2008; 58:1788–1791. PubMed http://dx.doi.org/10.1099/ijs.0.65656-0View ArticlePubMedGoogle Scholar
  7. Downes J, Sutcliffe IC, Booth V, Wade WG. Prevotella maculosa sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2007; 57:2936–2939. PubMed http://dx.doi.org/10.1099/ijs.0.65281-0View ArticlePubMedGoogle Scholar
  8. Downes J, Liu M, Kononen E, Wade WG. Prevotella micans sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2009; 59:771–774. PubMed http://dx.doi.org/10.1099/ijs.0.002337-0View ArticlePubMedGoogle Scholar
  9. Hayashi H, Shibata K, Sakamoto M, Tomita S, Benno Y. Prevotella copri sp. nov. and Prevotella stercorea sp. nov., isolated from human faeces. Int J Syst Evol Microbiol 2007; 57:941–946. PubMed http://dx.doi.org/10.1099/ijs.0.64778-0View ArticlePubMedGoogle Scholar
  10. Lawson PA, Moore E, Falsen E. Prevotella amnii sp. nov., isolated from human amniotic fluid. Int J Syst Evol Microbiol 2008; 58:89–92. PubMed http://dx.doi.org/10.1099/ijs.0.65118-0View ArticlePubMedGoogle Scholar
  11. Alauzet C, Mory F, Carlier JP, Marchandin H, Jumas-Bilak E, Lozniewski A. Prevotella nanceiensis sp. nov., isolated from human clinical samples. Int J Syst Evol Microbiol 2007; 57:2216–2220. PubMed http://dx.doi.org/10.1099/ijs.0.65173-0View ArticlePubMedGoogle Scholar
  12. Sakamoto M, Ohkusu K, Masaki T, Kako H, Ezaki T, Benno Y. Prevotella pleuritidis sp. nov., isolated from pleural fluid. Int J Syst Evol Microbiol 2007; 57:1725–1728. PubMed http://dx.doi.org/10.1099/ijs.0.64885-0View ArticlePubMedGoogle Scholar
  13. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 2011; 28:2731–2739. PubMed http://dx.doi.org/10.1093/molbev/msr121PubMed CentralView ArticlePubMedGoogle Scholar
  14. Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed http://dx.doi.org/10.1073/pnas.87.12.4576PubMed CentralView ArticlePubMedGoogle Scholar
  15. Validation List No. 143. Int J Syst Evol Microbiol 2012; 62:1–4. http://dx.doi.org/10.1099/ijs.0.039487-0
  16. Krieg NR, Ludwig W, Euzéby J, Whitman WB. Phylum XIV. Bacteroidetes phyl. nov. In: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ, Ward NL, Ludwig W, Whitman WB (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 4, Springer, New York, 2011Google Scholar
  17. Krieg NR. Class I. Bacteroidia class. nov. In: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ, Ward NL, Ludwig W, Whitman WB (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 4, Springer, New York, 2011Google Scholar
  18. Krieg NR. Order I. Bacteroidales ord. nov. In: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ, Ward NL, Ludwig W, Whitman WB (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 4, Springer, New York, 2011, p. 25.Google Scholar
  19. Krieg NR. Family V. Prevotellaceae fam. nov. In: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ, Ward NL, Ludwig W, Whitman WB (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 4, Springer, New York, 2011, p. 85.Google Scholar
  20. Shah HN, Collins DM. Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int J Syst Bacteriol 1990; 40:205–208. PubMed http://dx.doi.org/10.1099/00207713-40-2-205View ArticlePubMedGoogle Scholar
  21. Willems A, Collins MD. 16S rRNA gene similarities indicate that Hallella seregens (Moore and Moore) and Mitsuokella dentalis (Haapasalo et al.) are genealogically highly related and are members of the genus Prevotella: emended description of the genus Prevotella (Shah and Collins) and description of Prevotella dentalis comb. nov. Int J Syst Bacteriol 1995; 45:832–836. PubMed http://dx.doi.org/10.1099/00207713-45-4-832View ArticlePubMedGoogle Scholar
  22. Sakamoto M, Moriya Ohkuma M. Reclassification of Xylanibacter oryzae Ueki et al. 2006 as Prevotella oryzae comb. nov., with an emended description of the genus Prevotella. Int J Syst Evol Microbiol 2012; 62:2637–2642. PubMed http://dx.doi.org/10.1099/ijs.0.038638-0View ArticlePubMedGoogle Scholar
  23. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25–29. PubMed http://dx.doi.org/10.1038/75556PubMed CentralView ArticlePubMedGoogle Scholar
  24. Prodigal http://prodigal.ornl.gov/
  25. GenBank database. http://www.ncbi.nlm.nih.gov/genbank
  26. Tatusov RL, Galperin MY, Natale DA, Koonin EV. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res 2000; 28:33–36. PubMed http://dx.doi.org/10.1093/nar/28.1.33PubMed CentralView ArticlePubMedGoogle Scholar
  27. Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997; 25:955–964. PubMed http://dx.doi.org/10.1093/nar/25.5.0955PubMed CentralView ArticlePubMedGoogle Scholar
  28. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 2007; 35:3100–3108. PubMed http://dx.doi.org/10.1093/nar/gkm160PubMed CentralView ArticlePubMedGoogle Scholar
  29. Krogh A, Larsson B, von Heijni G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001; 305:567–580. PubMed http://dx.doi.org/10.1006/jmbi.2000.4315View ArticlePubMedGoogle Scholar
  30. Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004; 340:783–795. PubMed http://dx.doi.org/10.1016/j.jmb.2004.05.028View ArticlePubMedGoogle Scholar
  31. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, et al. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics 2008; 9:75–89. PubMed http://dx.doi.org/10.1186/1471-2164-9-75PubMed CentralView ArticlePubMedGoogle Scholar

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© The Author(s) 2014