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Alignment-Based Approach for Durable Data Storage into Living Organisms
Nozomu Yachie,†,‡ Kazuhide Sekiyama,†,‡ Junichi Sugahara,†,§ Yoshiaki Ohashi,*,†,| andMasaru Tomita†,‡,§,|
Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, 997-0017 Yamagata, Japan;
Bioinformatics Program, Graduate School of Media and Governance, Keio University, 5322 Endo, Fujisawa, 252-8520
Kanagawa, Japan; Department of Environmental Information, Keio University, 5322 Endo, Fujisawa, 252-8520 Kanagawa,Japan; and Human Metabolome Technologies, Inc., 246-2 Mizukami, Kakuganji, Tsuruoka, 997-0052 Yamagata, Japan

The practical realization of DNA data storage is a major scientific goal. Here we introduce a
simple, flexible, and robust data storage and retrieval method based on sequence alignment of
the genomic DNA of living organisms. Duplicated data encoded by different oligonucleotide
sequences was inserted redundantly into multiple loci of the Bacillus subtilis genome. Multiplealignment of the bit data sequences decoded by B. subtilis genome sequences enabled the retrievalof stable and compact data without the need for template DNA, parity checks, or error-correcting
algorithms. Combined with the computational simulation of data retrieval from mutated message
DNA, a practical use of this alignment-based method is discussed.

Introduction
DNA has often been proposed to be a reproducible, heritable
media in which a substantial amount of information can be
included in its nucleotide sequence, while paper, magnetic
media, and silicon chips are used for data storage today. Since
all of these media are easily destroyed by personal and naturalaccidents, they are in need of endless attention to maintain theircontents. Development of a DNA memory technology utilizing
living organisms is of much greater potential than any of theexisting counterparts to render a service of inheriting data. The
robustness of DNA data, however, ensures the maintenance ofarchived information over extensive periods of time (hundreds
to thousands of years) (1-4).
Message DNA has been used in steganography (5), as ameans of short trademarks/signatures (6) and long-term storage(3, 7). For economical and secure data encoding, sophisticateddesigns of DNA message regions and template primer sequences
based on the polymerase chain reaction (PCR) method havebeen reported for data retrieval (4, 8). Various codes have beendeveloped for encryption in DNA sequences, including the
economical Huffman code based on Huffman’s algorithm thatis used for short-term data storage (4). Similar to the parity-bit
operation within barcodes of goods, the comma code uses asingle base to punctuate the message and create an automatic
DNA reading frame, while the redundant alternate code
comprises an alternating sequence of purines and pyrimidines;
these codes utilize DNA durability to archive long-term data(4). Retrieval of these forms of data requires PCR-based* To whom correspondence should be addressed. Tel: +81-235-29-0526.
Fax: +81-235-29-0529. E-mail: ohashi@sfc.keio.ac.jp.
† Institute for Advanced Biosciences, Keio University.
‡ Graduate School of Media and Governance, Keio University.
§ Department of Environmental Information, Keio University.
| Human Metabolome Technologies, Inc.
10.1021/bp060261y CCC: $37.00

amplification, using template primer sequences, and DNA
sequencing. In order to avoid misreading caused by wobble
DNA annealing, a comma-free DNA design with attention totemplate DNA was proposed (8). However, these codes are notfully essential for the encoded DNA region to offer somesafekeeping against mutation, which might make alternations
to the sense of encoded data (4). Degradation of data could occur
during DNA preparation in the laboratory, during storage, or atdata retrieval (4). Additionally, genetic evolution and adaptationof the host organism could lead to the accumulation of mutations
such as point mutations, insertions, and deletions in chromosomal DNA, resulting in destruction of data inheritance.
Magnetic disk drives store data securely by duplicating
redundant data or parity codes into different sectors; data arethen retrieved by the assembly of partial sectors. Here, we
propose a method to copy and paste data within the genomic
sequence of a living organism, Bacillus subtilis, thus acquiring
versatile data storage and the robustness of data inheritance.
Two or more different features of oligonucleotide DNA
molecules compressed from the same data with an economicalcode were duplicated into multiple genomic loci for data storage.
The encoded data is then retrievable by complete genome
sequencing and searching for duplicated coding regions using
multiple alignments of all the possible decoded bit-data sequences of the genomic DNA. Data durability was further
discussed and estimated by computational simulation. We
propose that this alignment-based method will be most beneficial
in utilizing DNA as trademarks/signatures of living modified
organisms (LMOs) and as valuable heritable media.

Materials and Methods
Conversion of Messages into Genetic Sequences. We
encoded the message “E)mc∧2 1905!” into the B. subtilis
genome. The message was initially converted into binary© 2007 American Chemical Society and American Institute of Chemical Engineers
Published on Web 01/25/2007

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Figure 1. Encryption of a message in the B. subtilis genome. (A) The message “E)mc∧2 1905!”, including the “shift” and “space” keys, wasconverted into hexadecimal code (separated by %) according to the Keyboard Scan Code (Set2). The binary code was then produced by four-bit
representation of each hexadecimal number. (B) The encryption key used to encode the message in DNA. Each pattern of four-bit binary code isrepresented by dinucleotides. (C) DNA cassettes C1, C2, C3, and C4 were translated by one-bit by one-bit frame shifting. Two sets of dinucleotides
at the 5′ and 3′ regions encode different binary codes (see Table 1 for details). (D) Cassettes were subcloned into pHASH202 or pHASH203
plasmids and inserted into the B. subtilis genome.

sequence according to the make signals of the Keyboard Scan
Code Set2 (9) that represent all keyboard inputs (Figure 1A).
The hexadecimal codes generated from keyboard inputs were
shifted into bit data, and the encryption keys translated eachfour-bit binary code into dinucleotides (Figure 1B). Because
every encryption key has the reading frame size of four-bit, it
is possible to translate a bit data sequence into four multiple
oligonucleotide sequences in the four different reading frames(Figure 1C). In this study, four of all of the possible different
oligonucleotide cassettes, C1, C2, C3 and C4, were designed
for the data storage of the message “E)mc∧2 1905!” (Table
1).
Introduction of Data into Plasmid Vectors. Introductionof the message sequences, C1 to C4, was carried out assummarized in Figure 1D. The chemically synthesized oligonucleotides C1 and C2 were annealed and subcloned into theT-extended SmaI and PVuII sites, respectively, of pHASH202(pSPIBER01); C3 and C4 were cloned into the SmaI and PVuII
sites, respectively, of pHASH203 (pSPIBER02) (10). Escherichia coli strain DH5R was used as the cloning host. The
sequence of each DNA cassette was confirmed using the DNA
sequencer ABI3100 (Applied Bioscience, CA).

Transformation of B. subtilis. B. subtilis competent cells
were prepared and transformed by the two-step culture method(11). In order to introduce the cassettes, B. subtilis BEST2136
was used as a recipient (Table 2). This strain is resistant toerythromycin (Em), blasticidin S (BS), and tetracycline (Tc).
The GB01 cells were generated by a mixture of 0.1 µg mL-1
of BEST2136 genomic DNA and 1 µg mL-1 of the pSPIBER01
plasmid. The chloramphenicol (Cm)-resistant transformants wereselected on LB medium plates containing 5 µg mL-1 of Cm.
The colonies that appeared after incubation at 37 °C overnight
displayed an Em-sensitive and BS/Cm/Tc-resistant phenotype,indicating that the cassette C1 and C2 had been introduced
within the metB::pBREm locus in the genome. Then, the GB02
cells were generated by a mixture of 0.1 µg mL-1 of GB01
genomic DNA and 1 µg mL-1 of the pSPIBER02 plasmid. The
Em-resistant transformants were selected on LB medium plates
containing 5 µg mL-1 of Em. After overnight incubation at 37°C, the cells were BS-sensitive and Cm/Em/Tc-resistant,indicating that the cassettes C3 and C4 had been introduced
within the proB::pBRBS locus in the genome. The four
sequences of cassettes within the genomic DNA of the data-

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503

Table 1. Oligonucleotide Sequences Encoding “E)mc∧2 1905!”
cassette

oligonucleotide sequencea

encoded bit data

C1

AA,CA,GA,GA,AC,CA,GA,AC,GT,TA,GG,GA,
CA,CC,CC,CA,GT,GA,CG,CA,GC,AC,GC,AC,
CC,GA,GT,CA,GA,CA,GC,TT (64 nt)

0000,0001,0010,0010,0100,0001,0010,0100,1110,0011,1010,0010,0001,
0101,0101,0001,1110,0010,1001,0001,0110,0100,0110,0100,0101,0010,
1110,0001,0010,0001,0110,1111 (128 bit)

C2

GT,GA,AC,AC,AG,GA,AC,CG,AT,TC,AC,AC,
GA,GG,GG,TA,AT,CC,GA,GA,AT,AG,AT,AG,
GG,CC,AT,GA,AC,GA,AT (62 nt)

1110,0010,0100,0100,1000,0010,0100,1001,1100,0111,0100,0100,0010,
1010,1010,0011,1100,0101,0010,0010,1100,1000,1100,1000,1010,0101,
1100,0010,0100,0010,1100 (124 bit)

C3

AA,AC,AG,CG,AA,AC,CG,TA,AG,GT,AG,AG,
CC,CC,AC,TC,AG,GG,AC,CC,CG,CA,CG,CA,
AC,TG,AG,AC,AG,CC,AG (62 nt)

0000,0100,1000,1001,0000,0100,1001,0011,1000,1110,1000,1000,0101,
0101,0100,0111,1000,1010,0100,0101,1001,0001,1001,0001,0100,1011,
1000,0100,1000,0101,1000 (124 bit)

C4

AG,CG,CA,GA,AA,CG,GA,TC,CA,CT,CA,AA,
GG,GG,AG,TT,CA,AC,AG,TG,GA,TA,GA,GA,
CG,TC,AA,CG,AA,TG,TC (62 nt)

1000,1001,0001,0010,0000,1001,0010,0111,0001,1101,0001,0000,1010,
1010,1000,1111,0001,0100,1000,1011,0010,0011,0010,0010,1001,0111,
0000,1001,0000,1011,0111 (124 bit)a Nucleotide sequences are given in the 5′ f 3′ direction. Dinucleotides separated by commas indicate encryption keys transformed by 4-bit binary codes.
The 5′ and 3′ outer regions of bit data sequences (underlined) were designed differently to identify initiations and terminations of encoded data by sequencealignment.

Table 2. Bacterial Strains Usedstrainrelevant genotype

descriptiona

BEST2136
GB01
GB02

metB::pBREm proB::pBRBS
metB::cat::(C1, C2) proB::pBRBS
metB::cat::(C1, C2) proB::erm::(C3, C4)

recipient strain, Em/BS/Tc-resistantEm-sensitive, Cm/BS/Tc-resistant, with cassette C1 and C2
BS-sensitive, Em/Cm/Tc-resistant, with cassette C1, C2, C3, and C4

a BS, blasticidin S; Cm, chloramphenicol; Em, erythromycin; Tc, tetracycline.

encoded strain GB02 were confirmed by DNA sequencing(ABI3100, Applied Bioscience, CA).
Estimation of Data Durability by Computational Simulation. In order to estimate the durability of the data obtainedwith our method, we computationally simulated data retrievals
and calculated the data recovery rates from mutated and/or
deleted cassette sequences. We prepared 10,000 sets including
four pairs of different nucleotide cassettes and their corresponding bit data sequences harboring the same data region, varying
in length from 100 to 10,000 bits. The nucleotide sequenceswere randomly mutated and deleted of their partial fragments
in silico, and their corresponding bit data sequences werechanged. Then, in each set, the mutation/deletion rate of thetotal of the four cassette sequences was calculated, and the data
recovery rate was estimated; we calculated the percent of
positions where less than half of the bit characters were changedin the data region. Conducting the same procedure, the recovery
rates of the data encoded by two and three cassettes were
calculated.

Results and Discussion
The concept presented here is the use of sequence alignment
to retrieve encoded data that are duplicated in separate genomic
loci. For data storage, two or more sequences with different
oligonucleotide features are compressed and copied from thesame bit data through multiple data-to-nucleotide translations
and then inserted into the genomic DNA. Data retrieval depends
on the multiple alignment of all possible decoded bit data
sequences from the genomic DNA. According to alignmentbased data retrieval from the complete genome with multiple
inserted sequences, data durability, error-correcting, and parity
check operations are all supplemented without any otherqualified DNA code or template DNA design.
Data Retrieval by Complete Genome Sequencing. Using
this alignment-based approach, we demonstrated data storagein B. subtilis genomic DNA. For the secure retrieval of encoded
data, our stratagem does not require additional material suchas template DNA, only the sequencing of the complete genome.
Data retrieval conducted by PCR-based amplification using thetemplate DNAs designed for both the 5′- and 3′-regions of thesingle data region is vulnerable to the breakage of either of the
DNA annealing sites, which are essential even to read the partial
region of encoded data. Currently, a single DNA sequencer
requires a 24-h day to sequence the equivalent of a bacterial
genome, up to about two million bases (12). However, a growing
demand for greater speeds and lower costs is pushing thedevelopment of new sequence technologies, and it is likely thatnew machines will be capable of reading one million bases per
second in the relatively near future (12). This alignment-aided
data storage and retrieval hence will be better suited to practical
fast-lane genome sequencing techniques.
Preparation of Duplicated Data Cassettes. In order torecover data by sequence alignment, the length of data sequence
cannot be shorter than a certain length, as it can otherwise occur
by chance in the host genome. For instance, when a message
data is by two keyboard inputs, our encryption key method
creates an eight-nucleotide sequence within each cassette, which
may have several possible alignment patterns at the genomic
level. In such a case, to retrieve data securely we therefore needto add more redundant descriptions or contrive more complex
codes that make longer data regions in each cassette. Althoughmany bacteria species presumably contain a number of naturally
repeating sequences, synthetic sequences of more than 20
nucleotides is a sufficiently specific feature (p < 1 × 10-5) inthe 4.2 Mbp B. subtilis genomic DNA.
In order to prevent damage to genomic DNA during evolution, such as the deletion of junk DNA sequences especiallycaused by homologous recombination (13, 14), our methodology
introduced different nucleotide sequences encoding the samedata by multiple data compression paths. Construction of a large
piece of synthetic DNA is, however, still likely to be technicallydemanding and expensive. Although each nucleotide feature of
the duplicated data sequence differed from each other andredundant descriptions were required to encode data, the direct
data-to-nucleotide transitions maximized the information amountin each nucleotide cassette without any qualified DNA code,and the one-bit by one-bit frame shifting of the four-bit data
windows generated the lossless data copies. Therefore, the costof synthetic DNAs in this methodology is on a par with thoseof previous studies (4). The encryption keys of four-bit data to504

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Figure 2. DNA sequencing of the four cassettes inserted into the B. subtilis genome. The genome sequences around the cassettes C1, C2, C3, andC4 (boxed) are shown in A, B, C, and D, respectively. According to the encryption keys, all possible dinucleotides were decompressed to thefour-bit binary codes, which are displayed above each nucleotide sequence.

dinucleotides used in this study can replicate a maximum offour cassettes, but the number of cassettes is not limited by otherencryption keys. For example, the encryption keys of eight-bit
data to tetranucleotides have the potential to replicate data ineight cassettes.
Complementation of Durability by Alignment-Based Data
Assembly. For data retrieval, genome sequences with multiple
inserted nucleotide cassettes are decompressed to all possible
patterns of bit data sequences. In this study, the encryption keys
represent the one-to-one correspondence of dinucleotides andfour-bit binary codes, and two of the bit data sequences are
generated by a nucleotide sequence using two-bp sliding
windows with one-bp displacement; the total of four possible
bit data sequences are decompressed by the double-stranded
genome sequence. Without any template DNA and parity checkcode within the bounds of data sequences, the encoded dataand its sectional data breaks are recognizable by multiplealignments, searching conserved regions against all the decompressed bit data sequences. Point mismatch and gap spaceindicate mutations and partial deletions or insertions of genome
sequence, respectively. The alignment result provides a complemented complete data region and is achieved by the merger ofmultiple decompressed patterns from the nucleotide fragmentsof the inserted cassettes. It complements frameshifts and adjusts
encryption key reading frame misalignments. As such, it issimilar to TBLASTX, an alignment program utilizing geneticcodon rules that outputs possible amino acid sequences that arecomputationally translated from subjected nucleic acid sequences(15).
Although the multiplication of cassettes in this technique does
lead to redundant volumes, data are retrievable independentlyof any other error-correcting algorithms, resulting in theadvantage of data stability. Previous studies have revealed that
intricately intertwined parity effects within single data-coding
regions cost a certain volume of data sequence (4). The data
recovery rate is fragile and proportional to data breakage,
particularly that which occurs through long-range DNA deletion.
For the approximation of data stability, we computationallysimulated data retrievals from cassettes that were randomly
mutated and deleted (Figure 3). In this simulation result, threeor more cassettes were indicated to ensure a comparatively highrecovery rate. Over 99% of the encoded data was recoveredfrom four cassettes when their mutation/deletion rates were lessthan 15%. Moreover, the positions of the data breakages were
easily identified by the alignment results when these data could
not be rescued. The encoding of multiple data into separate
sections, much like the redundant arrays of inexpensive disk
(RAID) (16) technology frequently used in magnetic disk data
storage, undoubtedly contributes to the stability and durabilityof the data. In addition, the absence of boundary characteristics
from the template DNAs seen in previous studies enables theboundaries of the encoded sequences within genomic DNA toalso be identified by alignment.

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Institute for Advanced Biosciences, Keio University, for theircritical discussions, Dr. Kazuharu Arakawa for helpful suggestions on the preparation of the manuscript, and Professor
Mitsuhiro Itaya for providing B. subtilis strain BEST2136.

References and Notes

Figure 3. Expected data recovery rate. The solid lines represent the
average data recovery rate per head of mutation/deletion. Dotted linesand gray-filled areas indicate two-sided 95% confidence intervals.

Data Storage into the B. subtilis Genomic DNA. Datastorage into B. subtilis cells was performed by a two-step culture
method. B. subtilis BEST2136 was transformed using pSPIBER01, generating GB01 (Table 2). Sequentially, GB01 wastransformed using pSPIBER02, generating GB02 (Table 2). This
strain harbors C1 and C2 at the metB locus and C3 and C4 at
the proB locus. Insertion of the four cassettes was confirmed
by DNA sequencing (Figure 2). GB02 is available upon request.
Practical Use of Long-Term and Large-Volume Data
Storage. We have successfully demonstrated the data storageof the message “E)mc∧2 1905!” into the B. subtilis strain
BEST2136. One of the advantages of inserting data into
organisms is the realization of data inheritance. Although these
media vessels carry a significant risk of transmuting data intheir evolutionary processes, our simple alignment-based approach effectuates a higher durability of data inheritance. While
the size of the nucleotide sequence that can artificially be
inserted into the organism’s chromosomal DNA is limited (3),we suggest that B. subtilis is an acceptable species for large
volume data storage. Previous megacloning techniques havecloned the entire 3.5 Mb genome of Synechocystis PCC6803
into the 4.2 Mb genome of B. subtilis 168, resulting in a 7.7
Mb composite genome (17). Hence, adopting and developing
further codes and experimental methods or inserting plural
fragments into the partial volumes of multiple-species metagenomes will enable the storage of huge volumes of data inheritable media. We suggest that this simple, flexible, and robust
method offers a practical solution to data storage and retrieval
challenges in combination with other, previously publishedtechniques.

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Acknowledgment
This work was inspired by discussions with Dr. Masanori
Arita. The authors are grateful to the members of MGSP at theReceived August 28, 2006. Accepted December 18, 2006.
BP060261Y