Patent Description:
DNA has the capacity to hold vast amounts of information, readily stored for long periods in a compact form<NUM>,<NUM>. The idea of using DNA as a store for digital information has existed since <NUM><NUM>. Physical implementations of DNA storage have to date stored only trivial amounts of information - typically a few numbers or words of English text<NUM>-<NUM>. The inventors are unaware of large-scale storage and recovery of arbitrarily sized digital information encoded in physical DNA, rather than data storage on magnetic substrates or optical substrates.

Currently the synthesis of DNA is a specialized technology focused on biomedical applications. The cost of the DNA synthesis has been steadily decreasing over the past decade. It is interesting to speculate at what timescale data storage in a DNA molecule, as disclosed herein, would be more cost effective than the current long term archiving process of data storage on tape with rare but regular transfer to new media every <NUM> to <NUM> years. Current "off the shelf" technology for DNA synthesis equates to a price of around <NUM> bytes per U. Newer technology commercially available from Agilent Technologies (Santa Clara, CA) may substantially decrease this cost. However, account also needs to be made for regular transfer of data between tape media. The questions are both the costs for this transfer of data and whether this cost is fixed or diminishes over time. If a substantial amount of the cost is assumed to be fixed, then there is a time horizon at which use of DNA molecules for data storage is more cost effective than regular data storage on the tape media. After <NUM> years (at least <NUM> media transfers), it is possible that this data storage using DNA molecules is already cost effective.

A practical encoding-decoding procedure that stores more information than previously handled is described in this disclosure. The inventors have encoded five computer files - totaling <NUM> bytes (739kB) of hard disk storage and with an estimated Shannon information<NUM> of <NUM> × <NUM><NUM> bits - into a DNA code. The inventors subsequently synthesized this DNA, transported the synthesized DNA from the USA to Germany via the UK, sequenced the DNA and reconstructed all five computer files with <NUM>% accuracy.

The five computer files included an English language text (all <NUM> of Shakespeare's sonnets), a PDF document of a classic scientific paper<NUM>, a JPEG colour photograph and an MP3 format audio file containing <NUM> seconds of speech (from Martin Luther King's "I Have A Dream" speech). This data storage represents approximately <NUM> times as much information as the known previous DNA-based storage and covers a much greater variety of digital formats. The results demonstrate that DNA storage is increasingly realistic and could, in future, provide a cost-effective means of archiving digital information and may already be cost effective for low access, multi-decade archiving tasks.

The high capacity of DNA to store information stably under easily achieved conditions<NUM>,<NUM> has made DNA an attractive target for information storage since <NUM><NUM>. In addition to information density, DNA molecules have a proven track record as an information carrier, longevity of the DNA molecule is known and the fact that, as a basis of life on Earth, methods for manipulating, storing and reading the DNA molecule will remain the subject of continual technological innovation while there remains DNA-based intelligent life<NUM>,<NUM>. Data storage systems based on both living vector DNA<NUM>-<NUM> (in vivo DNA molecules) and on synthesized DNA<NUM>,<NUM> (in vitro DNA) have been proposed. The in vivo data storage systems have several disadvantages. Such disadvantages include constraints on the quantity, genomic elements and locations that can be manipulated without affecting viability of the DNA molecules in the living vector organisms. Examples of such living vector organisms include but are not limited to bacteria. The reduction in viability includes decreasing capacity and increasing the complexity of information encoding schemes. Furthermore, germline and somatic mutation will cause fidelity of the stored information and decoded information to be reduced over time and possibly a requirement for storage conditions of the living DNA to be carefully regulated.

In contrast, the "isolated DNA" (i.e., in vitro DNA) is more easily "written" and routine recovery of examples of the non-living DNA from samples that are tens of thousands of years old<NUM>-<NUM> indicates that a well-prepared non-living DNA sample should have an exceptionally long lifespan in easily-achieved low-maintenance environments (i.e. cold, dry and dark environments)<NUM>-<NUM>.

Previous work on the storage of information (also termed data) in the DNA has typically focused on "writing" a human-readable message into the DNA in encoded form, and then "reading" the encoded human-readable message by determining the sequence of the DNA and decoding the sequence. Work in the field of DNA computing has given rise to schemes that in principle permit large-scale associative (content-addressed) memory<NUM>,<NUM>-<NUM>, but there have been no attempts to develop this work as practical DNA-storage schemes. <FIG> shows the amounts of information successfully encoded and recovered in <NUM> previous studies (note the logarithmic scale on the y-axis). Points are shown for <NUM> previous experiments (open circles) and for the present disclosure (solid circle). The largest amount of human-readable messages stored this way is <NUM> characters of English language text<NUM>, equivalent to approximately <NUM> bits of Shannon information<NUM>.

The Indian Council of Scientific and Industrial Research has filed a U. Patent Application Publication No. <CIT>) that teaches a method for storing information in DNA. The method of U. '<NUM> comprises using an encoding method that uses <NUM>-DNA bases representing each character of an extended ASCII character set. A synthetic DNA molecule is then produced, which includes the digital information, an encryption key, and is flanked on each side by a primer sequence. Finally, the synthesized DNA is incorporated in a storage DNA. In the event that the amount of DNA is too large, then the information can be fragmented into a number of segments. The method disclosed in U. '<NUM> is able to reconstruct the fragmented DNA segments by matching up the header primer of one of the segments with the tail primer on the subsequent one of the segments.

International Patent Application <CIT> teaches a technique for using nucleic acid molecules a medium for long-term storage and retrieval of information. The method comprises synthesizing a so-called information nucleic acid molecule storing information as a nucleotide sequence and a so-called polyprimer key. The polyprimer key provides information necessary for amplification and sequence of the information nucleic acid molecule, thus permitting retrieval of the stored information. The information nucleic acids having the following sequence elements: common flanking forward and reverse PCTR amplification primers, a unique sequencing primer, a small common spacer serving as a cue to indicate the start of the store information, and a unique information segment. The information to be stored is encoded successively in these information segments, beginning with the first item of information. The polyprimer key is also flanked by the common forward and reverse PCR amplification primers and contains in the proper order the unique Seq Primers for the ordered retrieval from each of the information nucleic acid molecule of the corresponding information segment sequence.

Other patent publications are known which describe techniques for storing information in DNA. For example, <CIT> teaches a steganographic method for concealing coded messages in DNA. The method comprises concealing a DNA encoded message within a genomic DNA sample followed by further concealment of the DNA sample to a microdot. The application of this U. '<NUM> patent is in particular for the concealment of confidential information. Such information is generally of limited length and thus the document does not discuss how to store items of information that are of longer length. The same inventors have filed an International Patent Application published as International Publication No. <CIT>.

A method for storage of an item of information as a synthesized DNA segment according to claim <NUM> and a method for decoding the synthesized DNA segment to recover the item of information is disclosed. The method comprises encoding bytes in the item of information. The encoded bytes are represented using a representation schema by at least one DNA nucleotide to produce a DNA sequence in-silico. In a next step, the DNA sequence is split into a plurality of overlapping DNA segments of a length of <NUM> bases with an overlap of at least <NUM> bases. The plurality of overlapping DNA segments build in a redundancy of storage of the items of information. Indexing information is augmented to the plurality of overlapping DNA segments, wherein the indexing information specifies a unique location in the DNA sequence of any one of the overlapping DNA segments. The plurality of DNA segments with the augmented indexing information is synthesized to produce synthesized DNA segments. The synthesizing includes adding adapters to the DNA segments. Finally, the synthesized DNA segments are stored.

The addition of the indexing information to the DNA segments means that the position of the segments in the DNA sequence representing the item of information can be uniquely identified. There is no need to rely on a matching of a head primer with a tail primer. This makes it possible to recover almost the entire item of information, even if one of the segments has failed to reproduce correctly. If no indexing information were present, then there is a risk that it might not be possible to correctly reproduce the entire item of information if the segments could not be matched to each other due to "orphan" segments whose position in the DNA sequence cannot be clearly identified.

The use of the overlapping DNA segments means that a degree of redundancy is built into the storage of the items of information. If one of the DNA segments overlapping with one or more of the other DNA segment cannot be decoded, then the encoded bytes can still be recovered from the neighboring ones of the overlapping DNA segments. Redundancy is therefore built into the system.

Multiple copies of the DNA segments can be made using known DNA synthesis techniques. This provides an additional degree of redundancy to enable the item of information to be decoded, even if some of copies of the DNA segments are corrupted and cannot be decoded.

In one aspect of the invention, the representation schema used for encoding is designed such that adjacent ones of the DNA nucleotides are different. This is to increase the reliability of the synthesis, reproduction and sequencing (reading) of the DNA segments.

In a further aspect of the invention, a parity-check is added to the indexing information. This parity check enables erroneous synthesis, reproduction or sequencing of the DNA segments to be identified. The parity-check can be expanded to also include error correction information.

In one aspect of the invention, alternate ones of the synthesized DNA segments are reverse complemented. These provide an additional degree of redundancy in the DNA and means that there is more information available if any of the DNA segment is corrupted.

A method for decoding a synthesized DNA segment for readout of stored information is disclosed. The method comprises sequencing DNA segments synthesized by the method for storage of an item of information. The method further comprises identifying the indexing information and the message nucleotides. From the indexing information and the message nucleotides, the DNA-encoded file is reconstructed.

One of the main challenges for a practical implementation of DNA storage to date has been the difficulty of creating long sequences of DNA to a specified design. The long sequences of DNA are required to store large data files, such as long text items and videos. It is also preferable to use an encoding with a plurality of copies of each designed DNA. Such redundancy guards against both encoding and decoding errors, as will be explained below. It is not cost-efficient to use a system based on individual long DNA chains to encode each (potentially large) message<NUM>. The inventors have developed a method that uses 'indexing' information associated with each one of the DNA segments to indicate the position of the DNA segment in a hypothetical longer DNA molecule that encodes the entire message.

The inventors used methods from code theory to enhance the recoverability of the encoded messages from the DNA segment, including forbidding DNA homopolymers (i.e. runs of more than one identical base) that are known to be associated with higher error rates in existing high throughput technologies. The inventors further incorporated a simple error-detecting component, analogous to a parity-check bit<NUM> into the indexing information in the code. More complex schemes, including but not limited to error-correcting codes<NUM> and, indeed, substantially any form of digital data security (e.g. RAID-based schemes<NUM>) currently employed in informatics, could be implemented in future developments of the DNA storage scheme<NUM>.

The inventors selected five computer files to be encoded as a proof-of-concept for the DNA storage of this disclosure. Rather than restricting the files to human-readable information, files using a range of common formats were chosen. This demonstrated the ability of the teachings of the disclosure to store arbitrary types of digital information. The files contained all <NUM> of Shakespeare's sonnets (in TXT format), the complete text and figure of ref. <NUM> (in PDF format), a medium-resolution color photograph of the EMBL-European Bioinformatics Institute (JPEG <NUM> format), a <NUM> second extract from Martin Luther King's "I Have A Dream" speech (MP3 format) and a file defining the Huffman code used in this study to convert bytes to base-<NUM> digits (as a human-readable text file).

The five files selected for DNA-storage were as follows.

The five computer files comprise a total of <NUM> bytes, approximately equivalent to a Shannon information of <NUM> × <NUM><NUM> bits or <NUM> times as much encoded and recovered human-designed information as the previous maximum amount known to have been stored (see <FIG>).

The DNA encoding of each one of the computer files was computed using software and the method is illustrated in <FIG>. In one aspect of the invention <NUM> described herein, the bytes comprising each computer file <NUM> were represented in step <NUM> as a DNA sequence <NUM> with no homopolymers by an encoding scheme to produce an encoded file <NUM> that replaces each byte by five or six bases (see below) forming the DNA sequence <NUM>. The code used in the encoding scheme was constructed to permit a straightforward encoding that is close to the optimum information capacity for a run length-limited channel (i.e., no repeated nucleotides). It will, however, be appreciated that other encoding schemes may be used.

The resulting in silico DNA sequences <NUM> are too long to be readily produced by standard oligonucleotide synthesis. Each of the DNA sequences <NUM> was therefore split in step <NUM> into overlapping segments <NUM> of length <NUM> bases with an overlap of <NUM> bases. To reduce the risk of systematic synthesis errors introduced to any particular run of bases, alternate ones of the segments were then converted in step <NUM> to their reverse complement, meaning that each base is "written" four times, twice in each direction. Each segment was then augmented in step <NUM> with an indexing information <NUM> that permitted determination of the computer file from which the segment <NUM> originated and its location within that computer file <NUM>, plus simple error-detection information. This indexing information <NUM> was also encoded in step <NUM> as non-repeating DNA nucleotides, and appended in step <NUM> to the <NUM> information storage bases of the DNA segments <NUM>.

In total, all of the five computer files were represented by <NUM> strings of DNA. Each one of the strings of DNA comprised <NUM> nucleotides (encoding original digital information plus indexing information). The encoding scheme used had various features of the synthesized DNA (e.g. uniform segment lengths, absence of homopolymers) that made it obvious that the synthesized DNA did not have a natural (biological) origin. It is therefore obvious that the synthesized DNA has a deliberate design and encoded information<NUM>.

As noted above, other encoding schemes for the DNA segments <NUM> could be used, for example to provide enhanced error-correcting properties. It would also be straightforward to increase the amount of indexing information in order to allow more or larger files to be encoded. It has been suggested that the Nested Primer Molecular Memory (NPMM) scheme<NUM> reaches its practical maximum capacity at <NUM> unique addresses<NUM>, and there appears to be no reason why the method of the disclosure could not be extended beyond this to enable the encoding of almost arbitrarily large amounts of information.

One extension to the coding scheme in order to avoid systematic patterns in the DNA segments <NUM> would be to add change the information. Two ways of doing this were tried. A first way involved the "shuffling" of information in the DNA segments <NUM>, The information can be retrieved if one knows the pattern of shuffling. In one aspect of the disclosure different patterns of shuffles were used for different ones of the DNA segments <NUM>.

A further way is to add a degree of randomness into the information in each one of the DNA segments <NUM>. A series of random digits can be used for this, using modular addition of the series of random digits and the digits comprising the information encoded in the DNA segments <NUM>. The information can easily be retrieved by modular subtraction during decoding if one knows the series of random digits used. In one aspect of the disclosure, different series of random digits were used for different ones of the DNA segments <NUM>.

The digital information encoding in step <NUM> was carried out as follows. The five computer files <NUM> of digital information (represented in <FIG>) stored on a hard-disk drive were encoded using software. Each byte of each one of the five computer files <NUM> to be encoded in step <NUM> was represented as a sequence of DNA bases via base-<NUM> digits ('trits' <NUM>, <NUM> and <NUM>) using a purpose-designed Huffman code listed in Table <NUM> (below) to produce the encoded file <NUM>. This exemplary coding scheme is shown in outline in <FIG>. Each of the <NUM> possible bytes was represented by five or six trits. Subsequently, each one of the trits was encoded as a DNA nucleotide <NUM> selected from the three nucleotides different from the previous nucleotide (<FIG>). In other words, in the encoding scheme chosen for this aspect of the disclosure, each one of the three nucleotides was different from the previous one used to ensure no homopolymers. The resulting DNA sequence <NUM> was split in step <NUM> to DNA segments <NUM> of length <NUM> bases, as shown in <FIG>. Each one of the DNA segments overlapped the previous DNA segment by <NUM> bases, to give DNA segments of a length that was readily synthesized and to provide redundancy. Alternate ones of the DNA segments were reverse complemented.

The indexing information <NUM> comprised two trits for file identification (permitting <NUM><NUM> = <NUM> files to be distinguished, in this implementation), <NUM> trits for intra-file location information (permitting <NUM><NUM> = <NUM> locations per file) and one 'parity-check' trit. The indexing information <NUM> was encoded in step <NUM> as non-repeating DNA nucleotides and was appended in step <NUM> to the <NUM> information storage bases. Each indexed DNA segment <NUM> had one further base added in step <NUM> at each end, consistent with the 'no homopolymers' rule, that would indicate whether the entire DNA segment <NUM> were reverse complemented during the 'reading' stage of the experiment.

In total, the five computer files <NUM> were represented by <NUM> strings of DNA, each comprising <NUM> (<NUM> + <NUM> + <NUM> + <NUM> + <NUM> + <NUM>) nucleotides (encoding original digital information and indexing information).

The data-encoding component of each string in the aspect of the invention described herein can contain Shannon information at <NUM> bits per DNA base, which is close to the theoretical optimum of <NUM> bits per DNA base for base-<NUM> channels with run length limited to one. The indexing implementation <NUM> permits <NUM><NUM> = <NUM> unique data locations. Increasing the number of indexing trits (and therefore bases) used to specify file and intra-file location by just two, to <NUM>, gives <NUM><NUM> = <NUM> unique locations, in excess of the <NUM> that is the practical maximum for the NPMM scheme<NUM>,<NUM>.

The DNA synthesis process of step <NUM> was also used to incorporate 33bp adapters to each end of each one of the oligonucleotides (oligo) to facilitate sequencing on Illumina sequencing platforms:.

The <NUM> DNA segment designs <NUM> were synthesized in step <NUM> in three distinct runs (with the DNA segments <NUM> randomly assigned to runs) using an updated version of Agilent Technologies' OLS (Oligo Library Synthesis) process described previously<NUM>, <NUM> to create approx. 2x10<NUM> copies of each DNA segment design. Errors were seen to occur in only about one error per <NUM> bases and independently in different copies of the DNA segments <NUM>. Agilent Technologies adapted the phosphoramidite chemistry developed previously<NUM> and employed inkjet printing and flow cell reactor technologies in Agilent's SurePrint in situ microarray synthesis platform. The inkjet printing within an anhydrous chamber allows the delivery of very small volumes of phosphoramidites to a confined coupling area on a 2D planar surface, resulting in the addition of hundreds of thousands of bases in parallel. Subsequent oxidation and detritylation are carried out in a flow cell reactor. Once the DNA synthesis has been completed, the oligonucleotides are then cleaved from the surface and deprotected<NUM>.

The adapters were added to the DNA segments to enable a plurality of copies of the DNA segments to be easily made. A DNA segment with no adapter would require additional chemical processes to "kick start" the chemistry for the synthesis of the multiple copies by adding additional groups onto the ends of the DNA segments.

Up to ~<NUM>% coupling efficiency is achieved by using thousands-fold excess of phosphoramidite and activator solution. Similarly, millions-fold excess of detritylation agent drives the removal of the <NUM>'-hydroxyl protecting group to near completion. A controlled process in the flowcell reactor significantly reduced depurination, which is the most prevalent side reaction<NUM>. Up to <NUM> unique sequences can be synthesized in parallel and delivered as ~<NUM>-<NUM> picomole pools of oligos.

The three samples of lyophilized oligos were incubated in Tris buffer overnight at <NUM>, periodically mixed by pipette and vortexing, and finally incubated at <NUM> for <NUM> hour, to a concentration of 5ng/ml. As insolubilized material remained, the samples were left for a further <NUM> days at <NUM> with mixing two-four times each day. The samples were then incubated at <NUM> for <NUM> hour and <NUM> for <NUM> minutes, and purified from residual synthesis byproducts on Ampure XP paramagnetic beads (Beckman Coulter) and could be stored in step <NUM>. Sequencing and decoding is shown in <FIG>.

The combined oligo sample was amplified in step <NUM> (<NUM> PCR cycles using thermocycler conditions designed to give even A/T vs. G/C processing<NUM>) using paired-end Illumina PCR primers and high-fidelity AccuPrime reagents (Invitrogen), a combination of Taq and Pyrococcus polymerases with a thermostable accessory protein. The amplified products were bead purified and quantified on an Agilent <NUM> Bioanalyzer, and sequenced using AYB software in paired-end mode on an Illumina HiSeq <NUM> to produce reads of <NUM> bases.

The digital information decoding was carried out as follows. The central <NUM> bases of each oligo were sequenced in step <NUM> from both ends and so rapid computation of full-length (<NUM> base) oligos and removal of sequence reads inconsistent with the designs was straightforward. The sequence reads were decoded in step <NUM> using computer software that exactly reverses the encoding process. The sequence reads for which the parity-check trit indicated an error or that at any stage could not be unambiguously decoded or assigned to a reconstructed computer file were discarded in step <NUM> from further consideration.

The vast majority of locations within every decoded file were detected in multiple different sequenced DNA oligos, and simple majority voting in step <NUM> was used to resolve any discrepancies caused by the DNA synthesis or the sequencing errors. On completion of this procedure <NUM>, four of the five original computer files <NUM> were reconstructed perfectly. The fifth computer file required manual intervention to correct two regions each of <NUM> bases that were not recovered from any sequenced read.

During decoding in step <NUM>, it was noticed that one file (ultimately determined to be watsoncrick. pdf) reconstructed in silico at the level of DNA (prior to decoding, via base-<NUM>, to bytes) contained two regions of <NUM> bases that were not recovered from any one of the sequenced oligos. Given the overlapping segment structure of the encoding, each region indicated the failure of four consecutive segments to be synthesized or sequenced, as any one of four consecutive overlapping segments would have contained bases corresponding to this location. Inspection of the two regions indicated that the non-detected bases fell within long repeats of the following <NUM>-base motif:
<NUM>' GAGCATCTGCAGATGCTCAT <NUM>'.

It was noticed that repeats of this motif have a self-reverse complementary pattern. These are shown in <FIG>.

It is possible that long, self-reverse complementary DNA segments might not be readily sequenced using the Illumina paired-end process, owing to the possibility that the DNA segments might form internal nonlinear stem-loop structures that would inhibit the sequencing-by-synthesis reaction used in the protocol used in the method described in this document. Consequently, the in silico DNA sequence was modified to repair the repeating motif pattern and then subjected to subsequent decoding steps. No further problems were encountered, and the final decoded file matched perfectly the file watsoncrick. A code that ensured that no long self-complementary regions existed in any of the designed DNA segments could be used in future.

Table <NUM> shows an example of the exemplary Huffman coding scheme used to convert byte values (<NUM>-<NUM>) to base-<NUM>. For highly compressed information, each byte value should appear equally frequently and the mean number of trits per byte will be (<NUM>*<NUM> + <NUM>*<NUM>)/<NUM> = <NUM>. The theoretical maximum number of trits per byte is log(<NUM>)/log(<NUM>) = <NUM>.

The arbitrary computer file <NUM> is represented as a string SØ of bytes (often interpreted as a number between Ø and <NUM><NUM> - <NUM>, i.e. a value in the set {<NUM>. The string SØ is encoded using the Huffman code and converting to base-<NUM>. This generates a string S<NUM> of characters as the trit {Ø, <NUM>, <NUM>}.

Let us now write len() for the function that computes the length (in characters) of the string S<NUM>, and define n=len(S<NUM>). Represent n in base-<NUM> and prepend <NUM> to generate a string S<NUM> of trits such that len(S<NUM>)=<NUM>. Form the string concatenation S<NUM> = S<NUM>. S<NUM>, where S<NUM> is a string of at most <NUM> zeros is chosen so that len(S<NUM>) is an integer multiple of <NUM>.

S<NUM> is converted to the DNA string S<NUM> of characters in {A, C, G, T} with no repeated nucleotides (nt) using the scheme illustrated in the table below. The first trit of S4 is coded using the 'A' row of the table. For each subsequent trit, characters are taken from the row defined by the previous character conversion.

For each trit t to be encoded, select the row labeled with the previous nucleotide <MAT> used and the column labeled t and encode using the nt in the corresponding table cell.

Define N = len (S<NUM>), and let ID be a <NUM>-trit string identifying the original file and unique within a given experiment (permitting mixing of DNA form different files SØ in one experiment. Split S<NUM> into the overlapping DNA segments <NUM> of length <NUM> nt, each of the DNA segments <NUM> being offset from the previous one of the DNA segments <NUM> by <NUM> nt. This means there will be ((N/<NUM>)-<NUM>) DNA segments <NUM>, conveniently indexed i = Ø. (N/<NUM>)-<NUM>. The DNA segment i is denoted Fi and contains (DNA) characters 25i. <NUM>i+<NUM> ofS<NUM>.

Each DNA segment Fi is further processed as follows:
If i is odd, reverse complement the DNA segment Fi.

Let i3 be the base-<NUM> representation of i, appending enough leading zeros so that len(i3) = <NUM>. Compute P as the sum (mod <NUM>) of the odd-positioned trits in ID and i3, i.e. ID<NUM> + i3<NUM> + i3<NUM> + i3<NUM> + i3<NUM> + i3<NUM> + i3<NUM>. (P acts a 'parity trit' - analogous to a parity bit - to check for errors in the encoded information about ID and i.

Form the indexing information <NUM> string IX = ID. P (comprising <NUM>+<NUM>+<NUM> = <NUM> trits). Append the DNA-encoded (step <NUM>) version of IX to Fi using the same strategy as shown in the above table, starting with the code table row defined by the last character of Fi, to give indexed segment F'i.

Form F"i by prepending A or T and appending C or G to F'i -choosing between A and T, and between C and G, randomly if possible but always such that there are no repeated nucleotides. This ensures that one can distinguish a DNA segment <NUM> that has been reverse complemented (step <NUM>) during DNA sequencing from one that has not. The former will start with G|C and the end with T|A; the latter will start A|T and end C|G.

The segments F"I are synthesized in step <NUM> as actual DNA oligonucleotides and stored in step <NUM> and may be supplied for sequencing in step <NUM>.

Decoding is simply reverse of the encoding in step <NUM>, starting with the sequenced DNA segments <NUM> F"I of length <NUM> nucleotides. Reverse complementation during the DNA sequencing procedure (e.g. during PCR reactions) can be identified for subsequent reversal by observing whether fragments start with A|T and end with C|G, or start with G|C and end T|A. With these two 'orientation' nucleotides removed, the remaining <NUM> nucleotide of each DNA segment <NUM> can be split into the first <NUM> 'message' nucleotides and the remaining fifteen 'indexing information <NUM>' nucleotides. The indexing information nucleotide <NUM> can be decoded to determine the file identifier ID and the position index i3 and hence i, and errors may be detected by testing the parity trit P. Position indexing information <NUM> permits the reconstruction of the DNA-encoded file <NUM>, which can then be converted to base-<NUM> using the reverse of the encoding table above and then to the original bytes using the given Huffman code.

The DNA storage has different properties from the traditional tape-based storage or disk-based storage. The ~750kB of information in this example was synthesized in 10pmol of DNA, giving an information storage density of approximately one Tera-byte/gram. The DNA storage requires no power and remains (potentially) viable for thousands of years even by conservative estimates.

DNA Archives can also be copied in a massively parallel manner by the application of PCR to the primer pairs, followed by aliquoting (splitting) the resulting DNA solution. In the practical demonstration of this technology in the sequencing process this procedure was done multiple times, but this could also be used explicitly for copying at large scale the information and then physically sending this information to two or more locations. The storage of the information in multiple locations would provide further robustness to any archiving scheme, and might be useful in itself for very large scale data copying operations between facilities.

The decoding bandwidth in this example was at <NUM> bits/second, compared to disk (approximately one Terabit/second) or tape (<NUM> Megabit/second), and latency is also high (~<NUM> days in this example). It is expected that future sequencing technologies are likely to improve both these factors.

Modeling the full cost of archiving using either the DNA-storage of this disclosure or the tape storage shows that the key parameters are the frequency and fixed costs of transitioning between tape storage technologies and media. <FIG> shows the timescales for which DNA-storage is cost-effective. The upper bold curve indicates the break-even time (x-axis) beyond which the DNA storage as taught in this disclosure is less expensive than tape. This assumes that the tape archive has to be read and re-written every <NUM> years (f = <NUM>/<NUM>), and depends on the relative cost of DNA-storage synthesis and tape transfer fixed costs (y-axis). The lower bold curve corresponds to tape transfers every <NUM> years. The region below the lower bold curve indicates cases for which the DNA storage is cost-effective when transfers occur more frequently than every <NUM> years; between the two bold curves, the DNA storage is cost-effective when transfers occur from <NUM>- to <NUM>-yearly; and above the upper bold curve tape is less expensive when transfers occur less frequently than every <NUM> years. The dotted horizontal lines indicate ranges of relative costs of DNA synthesis to tape transfer of <NUM>-<NUM> (current values) and <NUM>-<NUM> (achieved if DNA synthesis costs reduce by an order of magnitude). Dotted vertical lines indicate corresponding break-even times. Note the logarithmic scales on all axes.

One issue for long-term digital archiving is how DNA-based storage scales to larger applications. The number of bases of the synthesized DNA needed to encode the information grows linearly with the amount of information to be stored. One must also consider the indexing information required to reconstruct full-length files from the short DNA segments <NUM>. The indexing information <NUM> grows only as the logarithm of the number of DNA segments <NUM> to be indexed. The total amount of synthesized DNA required grows sub-linearly. Increasingly large parts of each ones of the DNA segments <NUM> are needed for indexing however and, although it is reasonable to expect synthesis of longer strings to be possible in future, the behavior of the scheme was modeled under the conservative constraint of a constant <NUM> nucleotides available for both the data and the indexing information <NUM>.

As the total amount of information increases, the encoding efficiency decreases only slowly (<FIG>). In the experiment (megabyte scale) the encoding scheme is <NUM>% efficient. <FIG> indicates that efficiency remains ><NUM>% for data storage on petabyte (PB, <NUM><NUM> bytes) scales and ><NUM>% on exabyte (EB, <NUM><NUM> bytes) scales, and that DNA-based storage remains feasible on scales many orders of magnitude greater than current global data volumes. <FIG> also shows that costs (per unit information stored) rise only slowly as data volumes increase over many orders of magnitude. Efficiency and costs scale even more favourably if we consider the lengths of the synthesized DNA segments <NUM> available using the latest technology. As the amount of information stored increases, decoding requires more oligos to be sequenced. A fixed decoding expenditure per byte of encoded information would mean that each base is read fewer times and so is more likely to suffer decoding error. Extension of the scaling analysis to model the influence of reduced sequencing coverage on the per-decoded-base error rate revealed that error rates increase only very slowly as the amount of information encoded increases to a global data scale and beyond. This also suggests that the mean sequencing coverage of <NUM>,<NUM> times was considerably in excess of that needed for reliable decoding. This was confirmed by subsampling from the <NUM>. 6x310<NUM> read-pairs to simulate experiments with lower coverage.

<FIG> indicates that reducing the coverage by a factor of <NUM> (or even more) would have led to unaltered decoding characteristics, which further illustrates the robustness of the DNA-storage method. Applications of the DNA-based storage might already be economically viable for long-horizon archives with a low expectation of extensive access, such as government and historical records. An example in a scientific context is CERN's CASTOR system, which stores a total of <NUM> PB of Large Hadron Collider data and grows at <NUM> PB yr-<NUM>. Only <NUM>% is maintained on disk, and CASTOR migrates regularly between magnetic tape formats. Archives of older data are needed for potential future verification of events, but access rates decrease considerably <NUM>-<NUM> years after collection. Further examples are found in astronomy, medicine and interplanetary exploration.

<FIG> shows the encoding efficiency and costs change as the amount of stored information increases. The x-axis (logarithmic scale) represents the total amount of information to be encoded. Common data scales are indicated, including the three zettabyte (<NUM> ZB, 3x10<NUM> bytes) global data estimate. The y-axis scale to left indicates encoding efficiency, measured as the proportion of synthesized bases available for data encoding. The y-axis scale to right indicate the corresponding effect on encoding costs, both at current synthesis cost levels (solid line) and in the case of a two-order-of magnitude reduction (dashed line).

<FIG> shows per-recovered-base error rate (y-axis) as a function of sequencing coverage, represented by the percentage of the original <NUM>. 6x10<NUM> read-pairs sampled (x axis; logarithmic scale). One curve represents the four files recovered without human intervention: the error is zero when ≥<NUM>% of the original reads are used. Another curve is obtained by Monte Carlo simulation from our theoretical error rate model. The final curve represents the file (watsoncrick. pdf) that required manual correction: the minimum possible error rate is <NUM>%. The boxed area is shown magnified in the inset.

Claim 1:
A method for storage of an item of information (<NUM>) comprising:
- encoding (<NUM>) bytes of the item of information (<NUM>);
- representing, using a representation schema, the encoded (<NUM>) bytes by at least one DNA nucleotide to produce a DNA sequence (<NUM>);
- splitting (<NUM>) the DNA sequence (<NUM>) into a plurality of overlapping DNA segments (<NUM>) of a length of <NUM> bases with an overlap of at least <NUM> bases, wherein the plurality of overlapping DNA segments (<NUM>) build in a redundancy of storage of the items of information (<NUM>);
- augmenting (<NUM>) indexing information (<NUM>) to the plurality of overlapping DNA segments (<NUM>), the indexing information specifying a unique location in the DNA sequence (<NUM>) of any one of the overlapping DNA segments (<NUM>);
- synthesizing (<NUM>) the plurality of overlapping DNA segments (<NUM>) with the augmented indexing information to produce synthesized DNA segments (<NUM>), the synthesizing including adding adapters to the DNA segments (<NUM>); and
- storing (<NUM>) the synthesized DNA segments (<NUM>).