Gregory Carrier 2,6 , Sylvain Santoni 3 , Marguerite Rodier-Goud 4 ...

All protocols for Next Generation Sequencers (NGS) start
with the preparation of a DNA library from extracted DNA.
Quality, number, and length of the sequences produced by NGS
all depend on the quality of the library that was prepared, itself
dependent on the DNA quality. Generally, when using NGS,
one would wish to obtain the genomic information contained in
the nucleus without an excessive proportion of cytoplasmic
DNA. Multiple copies of cytoplasmic genomes are present in
every cell (11 to 70 copies, depending on the developmental stage
and/or physiological status) ( Tymms et al., 1983 ). The chloroplast
DNA of plants, therefore, represents 17 – 23% of their total
DNA ( Boffey and Leech, 1982 ).
In this study, we developed a protocol that strongly reduces
the amount of cytoplasmic DNA. The improvement in quality
1 Manuscript received 22 September 2010; revision accepted 24 October 2010.
This work was funded by the French Ministry of Research and Higher
education and the French Ministry of Food, Agriculture and Fisheries,
including a grant from the IFV for GC. We are grateful to Genotoul of
INRA Toulouse and Illinois University core facility for their help and advice
on NGS, and to Romain Guyot and Sylvie Faure for manuscript corrections.
We acknowledge Helen McCombie-Boudry for improving the English.
Authors ’ contributions: GC designed the protocol, carried out the nuclei
extraction, performed the grape 454 experiments and drafted the manuscript.
SS designed the experiment and helped to draft the manuscript. MRG
performed the microscope experiments. AC participated in the design of
the protocol. AdK and CDT provided the coffee material and analyzed
coffee 454 runs. JMB, PT, and LLC conceived and coordinated the study
and helped to draft the manuscript. All authors read, corrected, and approved
the fi nal manuscript.
6 Author for correspondence: gregory.carrier@supagro.inra.fr
doi:10.3732/ajb.1000371
AJB Advance Article published on December 23, 2010, as 10.3732/ajb.1000371.
The latest version is at http://www.amjbot.org/cgi/doi/10.3732/ajb.1000371
AJB Primer Notes & Protocols in the Plant Sciences
A N EFFICIENT AND RAPID PROTOCOL FOR PLANT NUCLEAR
DNA PREPARATION SUITABLE FOR NEXT GENERATION
SEQUENCING METHODS 1
American Journal of Botany: e1–e3, 2011; http://www.amjbot.org/ © 2011 Botanical Society of America
e1
American Journal of Botany: e000–e000. 2011.
Gregory Carrier 2,6 , Sylvain Santoni 3 , Marguerite Rodier-Goud 4 ,
Aur é lie Canaguier 5 , Alexandre de Kochko 3 , Christine Dubreuil-Tranchant
Patrice This 3 , Jean-Michel Boursiquot 3 , and Lo ï c Le Cunff 2
2 UMT Geno-Vigne, IFV-INRA-Montpellier Supagro, 2 place Viala, F-34060 Montpellier, France; 3 UMR 1097 DIAPC,
INRA-IRD-Montpellier Supagro, 2, place Viala, F-34060, Montpellier, France; 4 UMR 1098 DAP, CIRAD-INRA-Universit é
Montpellier II, Avenue Agropolis 34398 Montpellier France; and 5 UMR 1165 INRA-CNRS-Universit é d ’ Evry G é nomique
V é g é tale, 2 rue Gaston Cr é mieux 91057 Evry, France
Premise of the study : In this study, we developed a nuclear DNA extraction protocol for Next Generation Sequencers (NGS).
Methods and Results : We applied this extraction method to grapevines and coffee trees, which are known to contain many
secondary metabolites. The nuclear DNA obtained was sequenced by the 454/GS-FLX method. We obtained excellent results,
with less than 4% cytoplasmic DNA, in a similar way to a BAC (Bacterial Artifi cial Chromosome) – building protocol. We also
compared our protocol with a classic DNA extraction using specifi c cytoplasmic DNA amplifi cation. Results showed a lower
cytoplasmic DNA contamination with the new protocol.
Conclusions : The method presented here is fast and economical. The DNA obtained is of high quality, with a low level of
cytoplasmic DNA contamination, and very effi cient for the construction of sequencing libraries.
Key words: next generation sequencers; nuclear plant DNA extraction; nuclei isolation.
Copyright 2010 by the Botanical Society of America
makes the method perfectly adapted to library construction for
NGS. We applied this extraction method to the grapevine ( Vitis
vinifera L.) and coffee tree ( Coffea canephora Pierre ex A.
Froehner). These species are known to contain many secondary
metabolites, which can generate diffi culties for DNA extraction
( Peterson et al., 1997 ; Mattivi et al., 2006 ).
To establish the protocol, we were partially inspired by a
classic protocol for nuclear DNA plant extraction destined for
the construction of BAC (Bacterial Artifi cial Chromosome) libraries
( Peterson et al., 1997 ). The two stages of the protocol
are: (i) isolation of nuclei and (ii) nuclear DNA extraction. Nuclear
DNA obtained with this protocol was then sequenced using
454/GS-FLX technology (Roche, Basel, Switzerland) with
the Titanium kit.
MATERIALS AND METHODS
All details of different steps of protocol are accessible in the supplementary
methods (see Appendix S1).
For the fi rst step, we used the “ option Y ” described by Peterson et al. (2000)
with a small modifi cation for nuclei isolation. The quantity of DNA necessary
to build a sequencing library has to be between 2 and 5 µ g, and of a quality
equivalent to that necessary for BAC library construction. To obtain this DNA,
we started with 5 to 6 g of leaves. Nuclei extraction is performed by fi rst crushing
the leaves quickly with a mortar and pestle in liquid nitrogen, so that enough
intact nuclei are freed. A fi ne homogeneous powder is then obtained, which is
suspended in a sucrose-based buffer containing 2- β -Mercaptoethanol and PVP
to protect nuclei from oxidation. The pH is maintained relatively high to inhibit
nuclease activity. The addition of Triton X-100 degrades some of the chloroplasts
and mitochondria. This stage requires care, however, as it cannot be extended:
lengthening the incubation would also lead to destruction of the nuclei.
We used additional fi ltration steps compared with the protocol of Peterson
et al. (2000) . After fi ltration through Miracloth and all the centrifugations, a
3 ,

e2 American Journal of Botany [Vol. 0
Fig. 1. Picture of nuclei grapevine extract with in DAPI coloration. Two pictures taken using a fl uorescence microscope. Blue corresponds to DNA,
red to chlorophyll, and green to fragment wall. Intact nuclei (blue spheres) and a few chloroplasts (red spheres) are visible on this grapevine extraction.
fi ltration step is done with a cell strainer (d: 40 µ m). The different stages of slow
centrifugation allow the nuclei to be precipitated in the bottom of the tube,
whereas fragments, chloroplasts, and mitochondria remain at the surface and
are then eliminated by fi ltration. After nuclei isolation, we examined a drop of
the suspension under a fl uorescence microscope with DAPI. Figure 1 shows the
large numbers of intact nuclei present in this suspension. We compared the
quality of visible chloroplasts in DAPI before and after nuclei purifi cation and
noted a signifi cant reduction in chloroplast number.
The second step of the protocol was nuclear DNA extraction. The objective
of preparation for a BAC library is to remove high molecular weight nuclear
DNA. Nuclear DNA for NGS does not require this to be done because the DNA
used for NGS is fragmented during library preparation. DNA extraction of a
nuclei suspension can be made with a commercial kit such as DNeasy Plan
Maxi Kit from Qiagen (Hilden, Germany). In this study, we preferred to use a
protocol in which we had complete control of all the steps, to obtain DNA of
high quality. In this procedure, the nuclei suspension obtained is then treated by
proteinase K in the presence of lauryl sarkosyl detergent, which helps membrane
lysis and the denaturation of cytoskeletal proteins, thus permitting the
freeing of nuclear DNA. To purify the DNA, a precipitation with potassium
acetate (salting out) eliminates broken cells and a maximum of distorted proteins.
Specifi c absorption of DNA is realized on silica matrix ( Boom et al.,
1990 ). The fi nal impurities are then eliminated by a succession of several
washes. Purifi ed DNA is then cleared of any alien element and is in an optimal
condition for use and conservation.
The quantity of purifi ed nuclear DNA was measured by the PicoGreen
method ( Murakami and McCaman, 1999 ). For grapevine and coffee tree, 12 and
7 µ g of nuclear DNA were extracted, respectively, from 5 g of leaves of each
species. Protocols for DNA library construction for the 454/Titanium (Roche)
sequencing technology recommend using 2 to 5 µ g of DNA. The improved
protocol we present here is therefore perfectly adapted to these quantities.
We used 454/GS-FLX Titanium as the NGS methodology to test our protocol,
but it is suitable for any other sequencing method. The 454 runs were successful.
For grapevine two runs were made, and we obtained 988 669 and
1 052 396, sequences, with mean lengths of 350 and 360 bp, respectively. For
coffee tree, a total of six runs were made. On average 1 325 441 sequences per
run were obtained for a mean length of 383 bp. These excellent results, although
they also depend on the quality of the sequencing libraries, are mainly
due to a high standard of the purifi ed DNA.
Cytoplasmic reference sequences are not available for coffee tree. Therefore,
to quantify the level of contaminant cytoplasmic DNA sequenced, we
worked on the grapevine dataset only. A BLAST search was conducted on
grapevine reference sequences from the NCBI website (http://www.ncbi.nlm.
nih.gov, Vitis vinifera chloroplast: NC 007957; Vitis vinifera mitochondrion:
NC 007762). Out of all sequences produced in the tests of our protocol, we
found that, on average, 1.7% were chloroplastic-like and 2.0% were mitochondrial-like
(E-value < 1e-50 and percentage identities > 85%). These results
show an elimination of a large proportion of cytoplasmic DNA by our protocol.
To compare this protocol with standard DNA extraction (Qiagen DNeasy
plant ), we used primer sets that specifi cally amplifi ed the ATPi and RPS16 chloroplastic
genes ( Heinze, 2007 ) on four different concentrations of DNA (1 ng/ µ l,
0.1 ng/ µ l, 0.01 ng/ µ l and 0.001 ng/ µ l). Results clearly indicated no amplifi cation
from 0.1 ng/ µ l with our protocol, while amplifi cation was achieved at this
concentration using the classic DNA extraction method. There is no amplifi cation
from 0.01 ng/ µ l using the classic DNA extraction method ( Fig. 2 ) . This
level of cytoplasmic DNA is similar to those obtained using BAC-building protocols
( Noir et al., 2004 ; Zharkikh et al., 2008 ). Cytoplasmic DNA is still suffi
ciently present to be sequenced during an NGS sequencing run. This aspect
could be valuable for gaining access to genomic information about cytoplasmic
DNA in an NGS run, without having an excessive proportion of these
sequences.
Fig. 2. Estimation of the elimination of chloroplastic DNA using the
new method compared with a classic DNA extraction protocol. (A) Specifi c
amplifi cation of ATPi. (B) Specifi c amplifi cation of RPS16, from different
DNA concentrations: 1ng/ µ l, 0.1 ng/ µ l, 0.01 ng/ µ l, 0.001 ng/ µ l with DNA
classic extraction.

January 2010] AJB Primer Notes & Protocols – Plant nuclear DNA preparation for next sequencing methods
CONCLUSIONS
The protocol presented here is fast, economical (compared
with a purchased kit), and does not require the use of dangerous
products. It provides adequate quantities of high-quality nuclear
DNA, low in cytoplasmic contaminants, and effi cient for constructing
sequencing libraries. This protocol can be adapted for
DNA preparations from many other plants containing high levels
of phenolic compounds or polysaccharides, and it is suitable
for all deep-sequencing technologies.
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495 – 503 .
Heinze , B. 2007 . A database of PCR primers for the chloroplast genomes
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e3
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