Direct Genomic DNA Sequencing of the D1/D2 Region of 26S rDNA of Basidiomycota and Ascomycota:

No DNA Amplification Necessary for Cultured SamplesB.W. Jarvis1*, B.L. Wickes2, L.M. Hoffman1

1EPICENTRE Biotechnologies, Madison, WI and 2UT Health Sciences Ctr., San Antonio, TX

ABSTRACT

Background: Fungal genomes contain 50-150 identicalcopies of the ribosomal genes (rDNA), a higher copynumber than in bacterial genomes. Until now, phylogenetic typing of fungi from both single culturesand mixed populations involved DNA extraction, amplification of regions of rDNA or internal transcribedsequences (ITS), purification (or cloning) of the PCRproduct, and DNA sequencing. The PCR may introducesequence errors, or chimeric PCR products when multiple rDNA sequences are present. We wished to eliminate the PCR steps of the identification process byobtaining sequence directly from genomic DNA (gDNA)of single cultures. Methods: gDNA was extracted fromyeast, filamentous fungi, and mushrooms using theEPICENTRE MasterPureTM Yeast DNA Purification Kit,according to the manufacturer’s recommendations.From 100 to 400 ng of gDNA was sequenced in 1/8,1/4, 1/2 and 1X BigDye™ sequencing reactions (ABI,Foster City, CA) using the LROR primer (5’-ACC CGCTGA ACT TAA GC-3’). Reactions were ethanol precipi-tated and sequenced on an ABI 310 Genetic Analyzer.Results: Sequence was obtained with reactions assmall as 1/8 X (1 microliter of BigDye terminator premix), and with DNA quantities as low as 100 ng. Sequences obtained without PCR allowedunambiguous identification of yeasts (for example:Candida krusei), filamentous fungi (Aspergillus nidu-lans), and mushrooms (Coprinus comatus). Conclusion:PCR is unnecessary for sequencing the rDNA regionsof DNA from isolated fungal species. In clinical settingswhere the speed of fungal identification is crucial, thedirect sequencing of isolated gDNA could be beneficial.Elimination of PCR may reduce errors introducedthrough amplification with thermostable polymerases,and remove the need to sequence multiple PCR products from a single isolate.

INTRODUCTION

There are several methods available for taxomic classi-fication of clinically occurring fungi. Chief among theseare methods based on biochemical tests1 and thosebased on DNA sequence identity. Among the DNAsequencing methods are pyrosequencing2, PCR-basedtechnologies3 and RAPD (Random AmplifiedPolymorphic DNA4). We have found that directsequencing of genomic DNA purified with theMasterPureTM Yeast DNA Isolation Kit provides 9-foldlonger read lengths than pyrosequencing for only a 1hour increase in time. Genomic DNA sequencing canbe performed on standard capillary-based instrumentscommonly found in molecular biology laboratories. Incontrast, the PCR-based methods require several morehours of time to provide equivalent sequence readlengths (see Flow Chart), and pyrosequencing useshighly specialized equipment. Fungal identification bydirect genomic DNA sequencing centers on the highlyrepetitive rDNA tandem repeats, which in fungi arerepeated up to 200 times5,6. The multiple copies of thisregion provide enough template for direct sequencing.For location of the rDNA primers used in this study, see FIG 1.

METHODS

Genomic DNA (gDNA) was extracted from yeast, fila-mentous fungi, and mushrooms using the EPICENTREMasterPureTM Yeast DNA Purification Kit, according tothe manufacturer’s recommendations. From 100 to 400ng of gDNA was sequenced in 1/8, 1/4, 1/2 and 1XBigDye® sequencing reactions (ABI, Foster City, CA)using the LROR primer (5’-ACC CGC TGA ACT TAAGC-3’), NL-1 primer (5’-GCA TAT CAA TAA GCG GAGGAA AAG), NL-4 primer (5’-GGT CCG TGT TTC AAGACG G) and ITS4 primer (5’-TCC TCC GCT TAT TGATAT GC). Reactions were ethanol precipitated andsequenced on an ABI PRISM® 310 Genetic Analyzer.Optimal sequencing was obtained with 1/4 X reactionsand 100 ng of gDNA templates.

RESULTS CONCLUSIONS

• Direct sequencing of gDNA from fungal cultures is arapid, convenient and relatively inexpensive methodfor identifying medically significant fungi.

• The multiple copies (50-200) of fungal rDNA genesprovide enough homogeneous template for directsequencing without amplification.

• Sequencing with commonly available instrumentationyields read lengths 9-fold longer than those obtainedfrom typical pyrosequencing. Thus, the accuracy ofBLAST sequence alignments is much enhanced.

• Yeast, filamentous fungus and even mushroom genomic DNA, purified by a salt precipitation method,can be used in direct sequencing.

• The MasterPure™ Yeast DNA Purification Kit providesexcellent yields of high molecular weight DNA fromyeasts and filamentous fungi.

• The avoidance of the polymerase chain reaction may decrease costs associated with licensing and royalties.

REFERENCES

1. Ciardo, D.E. et al. (2006) J. Clin. Microbiol. 44:77.

2. Ahmadian A., Ehn, M. and Hober, S. (2006) Clin Chim Acta.363:83.

3. Van Burik, J-A. et al. (1998) J. Clin. Microbiol. 36:1169.

4. Aoki, F.H. et al.(1999) J. Clin. Microbiol. 37: 315.

5. Xu, J. et al. (2005) New Microbiol. 28:135.

6. Rustchenko, E.P., Curran, T.M., and Sherman, F. (1993) J. Bacteriol. 175: 7189.

7. Jin, J. et al. (2004) J. Clin. Microbiol. 42:4293.

F-016

3-4hrsCycle Sequencing Rx

30minLoading Process

1 hr/sampleAutomated Sequencer

5minSequence Analysis

3-4hrsCycle Sequencing Rx

30minLoading Process

1 hr/sampleAutomated Sequencer

5minSequence Analysis

30minPCR Cleanup 15

minPyrosequencing Prep

1hrPyrosequencing

5minData Analysis

30minElectrophoresis

Total Time - 4.5 hoursRead Length - 70 bp

PYROSEQUENCING

Total Time - 8.5 hoursRead Length - 600 bp

PCR-BASED

Total Time - 5.5 hoursRead Length - 600 bp

GENOMIC

1.5hoursAmplification

i199

0506

Direct Genomic DNA Sequencing of Fungi vs. Alternative PCR-Based Methods1

hourDNA Purification

Sample Identity[gDNA]ng/µl

PrimersLROR e value NL-1 e value NL-4 e value ITS4 e value

Cryptococcus neoformans 39 yes 4e-91* yes 0.0 yes 0.0 yes 0.0

Yarrowia (Candida) lipolytica 46 no yes 0.0 yes 4e-9 yes 2e-156

Candida tropicalis 43 no no yes 2e-32 yes 2e-29

Candida paropsilosis 34 yes 7e-110 no no yes 0.0

Cryptococcus laurentii 116 yes 0.0 no no yes 8e-100

Clavispora (Candida) lusitaniae 37 no no yes 2e-29 yes 3e-173

Cryptococcus neoformans 25 yes 3e-119 yes 0.0 yes 3e-96 yes 0.0

Aspergillus fumigatus 48 no yes 2e-63 yes 8e-14 yes 6e-61

Candida albicans 25 nd nd nd yes 0.0

Candida glabrata 20 nd nd nd yes 0.0

Coprinus comatus 50 nd nd nd yes 0.0

nd = not determined

*Expect Value“. . . E value describes the random background noise that exists for matches between sequences. For example, an E value of 1 assigned to a hit can beinterpreted as meaning that in a database of the current size one might expect to see 1 match with a similar score simply by chance. This means that thelower the E-value, or the closer it is to "0" the more "significant" the match is.” See http://www.ncbi.nlm.nih.gov/blast/blast_FAQs.shtml

0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000DNA, nanograms

MasterPure™ –Supplier Q –Supplier F –

MasterPure™ –Supplier Q –Supplier F –

C. albicans

S. cerevisiae

FIG 2. The MasterPure™ Yeast DNA Purification Kit gives higheryields of DNA than other kits. DNA was quantitated specifically withHoechst 33258 fluorescent dye, which gives minimal fluorescence with RNA.The data represent the average DNA yields determined by fluorometry fromtwo experiments with S. cerevisiae and C. albicans. The MasterPure Kit produced up to 17 times more DNA from C. albicans and 12 times more DNAfrom S. cerevisiae than other kits.

PrimerName

OligoLength

TmQuality

Assessment* % TemplatesSequenced

LROR 17 50° C 83 38

NL-1 24 62° C 93 50

NL-4 19 58° C 78 75

ITS4 20 56° C 92 100

* Primer Premier software

800-284-8474

Table 1. Primer Quality Chart.

Table 2. gDNA Seq. Outcome Chart.

FIG 1. rDNA repeat diagram.

I200

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NL-1

LROR

NL-4ITS4

D1/D2 Region

promoter

18S 5.8S 26S 5S

terminator

ITS1 ITS2

rDNA Repeat (9.1 kb)

5S

Cryptococcus neoformans