Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> The microdot is a means of concealing messages (steganography)1 that was developed by Professor Zapp and used by German spies in the Second World War to transmit secret information2. A microdot (“the enemy's masterpiece of espionage”2) was a greatly reduced photograph of a typewritten page that was pasted over a full stop in an innocuous letter2. We have taken the microdot a step further and developed a DNA-based, doubly steganographic technique for sending secret messages. A DNA-encoded message is first camouflaged within the enormous complexity of human genomic DNA and then further concealed by confining this sample to a microdot. <s> BIB001 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> In this digital age, the technology used for information storage is undergoing rapid advances. Data currently being stored in magnetic or optical media will probably become unrecoverable within a century or less, through the combined effects of hardware and software obsolescence and decay of the <s> BIB002 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> For very long-term storage and retrieval, encode information as artificial DNA strands and insert into living hosts. As vectors, bacteria, even some bugs and weeds, might be good for hundreds of millions of years. <s> BIB003 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> The time is approaching when information can be written into DNA. This tutorial work surveys the methods for designing code words using DNA, and proposes a simple code that avoids unwanted hybridization in the presence of shift and concatenation of DNA words and their complements. <s> BIB004 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> 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. Multiple alignment of the bit data sequences decoded by B. subtilis genome sequences enabled the retrieval of 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. <s> BIB005 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> BackgroundDNA-based watermarks are helpful tools to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. In silico analyses showed that in coding regions synonymous codons can be used to insert encrypted information into the genome of living organisms by using the DNA-Crypt algorithm.ResultsWe integrated an authenticating watermark in the Vam7 sequence. For our investigations we used a mutant Saccharomyces cerevisiae strain, called CG783, which has an amber mutation within the Vam7 sequence. The CG783 cells are unable to sporulate and in addition display an abnormal vacuolar morphology. Transformation of CG783 with pRS314 Vam7 leads to a phenotype very similar to the wildtype yeast strain CG781. The integrated watermark did not influence the function of Vam7 and the resulting phenotype of the CG783 cells transformed with pRS314 Vam7-TB shows no significant differences compared to the CG783 cells transformed with pRS314 Vam7.ConclusionFrom our experiments we conclude that the DNA watermarks produced by DNA-Crypt do not influence the translation from mRNA into protein. By analyzing the vacuolar morphology, growth rate and ability to sporulate we confirmed that the resulting Vam7 protein was functionally active. <s> BIB006 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> We developed a system to encode digital information in DNA polymers based on the partial restriction digest (PRD). Our encoding method relies on the length of the fragments obtained by the PRD rather than the actual content of the nucleotide sequence, thus eliminating the need for expensive sequencing machinery. In this letter, we report on the encoding of 12 bits of data in a DNA fragment of 110 nucleotides and the process of recovering the data. <s> BIB007 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> A DNA Memory with over 10 million (16.8 M) addresses was achieved. The data embedded into a unique address was correctly extracted through an addressing processes based on nested PCR. The limitation of the scaling-up of the proposed DNA memory is discussed by using a theoretical model based on combinatorial optimization with some experimental restrictions. The results reveal that the size of the address space of the DNA memory presented here may be close to the theoretical limit. The high-capacity DNA memory can be also used in cryptography (steganography) or DNA ink. In decoding process, multiple data with different addresses can be also simultaneously accessed by using the mixture of some address primers. <s> BIB008 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> Data-encoding synthetic DNA, inserted into the genome of a living organism, is thought to be more robust than the current media. Because the living genome is duplicated and copied into new generations, one of the merits of using DNA material is long-term data storage within heritable media. A disadvantage of this approach is that encoded data can be unexpectedly broken by mutation, deletion, and insertion of DNA, which occurs naturally during evolution and prolongation, or laboratory experiments. For this reason, several information theory-based approaches have been developed as an error check of broken DNA data in order to achieve data durability. These approaches cannot efficiently recover badly damaged data-encoding DNA. We recently developed a DNA data-storage approach based on the multiple sequence alignment method to achieve a high level of data durability. In this paper, we overview this technology and discuss strategies for optimal application of this approach. <s> BIB009 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> An improved Huffman coding method for information storage in DNA is described. The method entails the utilization of modified unambiguous base assignment that enables efficient coding of characters. A plasmid-based library with efficient and reliable information retrieval and assembly with uniquely designed primers is described. We illustrate our approach by synthesis of DNA that encodes text, images, and music, which could easily be retrieved by DNA sequencing using the specific primers. The method is simple and lends itself to automated information retrieval. <s> BIB010 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> This article surveys all known fields of network coding theory and leads the reader through the antecedents of the network coding theory to the most recent results, considering also information theory and matroid theory. By focusing on providing ideas and not formulas, this survey is both fitted for the taste of readers who are mathematically oriented and newcomers to the area. Additionally, this survey also includes an innovative and clear graph representation of the most prominent literature on network coding theory, its relevance and evolution from the very beginning till today. <s> BIB011 </s> Natural Data Storage: A Review on sending Information from now to then via Nature <s> Encoding data in DNA <s> Digital production, transmission and storage have revolutionized how we access and use information but have also made archiving an increasingly complex task that requires active, continuing maintenance of digital media. This challenge has focused some interest on DNA as an attractive target for information storage because of its capacity for high-density information encoding, longevity under easily achieved conditions and proven track record as an information bearer. Previous DNA-based information storage approaches have encoded only trivial amounts of information or were not amenable to scaling-up, and used no robust error-correction and lacked examination of their cost-efficiency for large-scale information archival. Here we describe a scalable method that can reliably store more information than has been handled before. We encoded computer files totalling 739 kilobytes of hard-disk storage and with an estimated Shannon information of 5.2 × 10(6) bits into a DNA code, synthesized this DNA, sequenced it and reconstructed the original files with 100% accuracy. Theoretical analysis indicates that our DNA-based storage scheme could be scaled far beyond current global information volumes and offers a realistic technology for large-scale, long-term and infrequently accessed digital archiving. In fact, current trends in technological advances are reducing DNA synthesis costs at a pace that should make our scheme cost-effective for sub-50-year archiving within a decade. <s> BIB012
Data encoded in DNA can be used for encryption BIB011 or long term storage. Based on the purpose, DNA can be embedded in non-coding DNA (nc-DNA) or protein coding DNA (pc-DNA) or synthetic DNA. One can represent each base pair by using 2 bits, which gives 4 different possibilities that can be mapped to 16 combinations of DNA base pairs ( for instance 00 → AT, 01 → GC, 10 → TA and 11 → CG). A single byte (or 8 bits) can represent 4 DNA base pairs. The entire diploid human genome can be represented in terms of bytes, as described: 6 * 10 9 base pairs/diploid genome x 1 byte/4 base pairs = 1.5 * 10 9 bytes or 1.5 Gigabytes  ]. If we want to calculate data that can be stored in human body with consideration of human body consisting of 100 trillion cells, we will have 150 Zettabytes approximately (150 * 10 12 * 10 9 bytes) data stored in the DNA of any human. In the 20 th century, many researcher have translated English text, mathematical equations BIB005 , latin text BIB007 and simple musical notations BIB010 ] to DNA using different DNA coding principles BIB003 ] BIB004 Ohashi 2004] [Skinner et al. 2007 ] BIB008 ] BIB006 . Following are the main encoding approaches proposed for DNA based information storage systems as shown in Figure 4 . [ Davis 1996 ], a small organism comprises of short piece of synthetic DNA used to encode visual icon in bacteria E.coli. Data encoding is done according to the molecular size of the bases. C is the smallest base assigned with 1, T→2, A→3 and G→4. Instead of numeric values, each nucleotide was assigned with phase structure like C→X, T→XX, A→XXX, G→XXXX. The encoding was done by placing the nucleotide at each repeated position of bits 0s and 1s. Nucleotides were placed according to number of repeated bits of 0s and 1s. For instance, 1001011 = CTCCT, 10101= CCCCC. Mirovenus created was inserted into bacterial host cell by using plasmids. Encoding scheme used was not accurate, efficient and DNA developed to store data is not uniquely decodable. In subsequent year, other form of DNA based data encoding named Genesis [Kac 1999] , an artwork of Eduardo Kac was introduced. He created artificial art gene that comprises of digital DNA by converting the lines from bible into Morse code. Morse code denoted by dot (.) and dash (-) was converted to nucleotides with the principle rule of converting dash (-) and dot (.) to T and C and replacing word space and letter space by A and G. This gene was then inserted into florescent E. Coli bacteria. Both these laid foundation for storing data in DNA. But it lacks efficiency, accuracy and better encoding and decoding methods. 2.1.2. PCR based encoding models. In Clelland encoding models BIB001 , microdots was used to cipher the data in human genomic DNA. Secret message was inserted between the PCR (Polymerase Chain reaction) forward and reverse primer sequences called template regions. The idea was to encode the characters by trivial assignment of DNA codon as encryption key to each characters and insert it in human genomic DNA. For total possible 64 codons, each English characters will be assigned codons and rest can be used to encode some of the symbols like dots and commas. This was carried out in vitro by combining the message DNA with the genomic DNA in the solution over a 16-point microdot printed on filter paper. Decoding was based on template regions using PCR amplification. Recipient must be aware of encryption key and primer sequences. Data stored was secured but major limitation was scalability of data encoded in the limited size of microdots, only 136 bits data was encoded by . Fig 1(a) PCR based encoding model is template based method that consist of forward and reverse primer between which the information is inserted. Decoding is done by using this primers as template by PCR amplification . Fig  1(b) is the type of error that is base flip or break point indicates by B, deletion denoted by D and insertion of base denoted by i. Figure 1 (c) demonstrates the alignment based encoding model. Here I DNA sequence data that is converted to four different DNA chunks C1, C2, C3 and C4 using encoding principle. DNA chunks are inserted at different loci in genome. For decoding (see fig. 1(d) ), genome is sequenced and multiple alignment of different loci of genome is done for duplicated information inserted in multiple copies of genomic DNA. Figure 1 (e) depicts encoding data in different plasmids in various location of bacterial genome. Data can be retrieved back by sequencing the index primers followed by sequencing of plasmid library. using this approach. To make it more accurate, Bancroft BIB002 proposed the concept of information DNA (iDNA) that comprises information and single poly primer key (PPK) along with forward and reverse primer and common 5-6 bases spacer to indicate the stored information. This concept resembles to the retrieval of information from an addressable storage device such as the random access memory in a computer where, PPK acts as data location identifiers. Data encoding was done by mapping ternary codes to only three bases A, C, and T but sequencing primers were designed with all four bases with the requirement that each fourth position be a G to prevent mispriming. Decoding can be done by sequencing PPK first to decode the forward and reverse primers and then based on specific sequencing primer one can retrieve the information. Total data encoded by using this approach was 561 bits. Drawbacks for PCR based methods are requirement of PCR, knowledge of primers and extensive experimental hurdles and practical issues. Moreover main drawback for PCR based methods are insertion of errors in template regions make the retrieval of encoded data impossible. 2.1.3. Alignment based encoding models. For the first time Yachie et al., BIB005 ] BIB009 introduced PCR independent alignment based data encryption using four bits per two bases encoding scheme. In this multiple sequence alignment based approach was used to encode the information into genomic DNA of B.subtilis. Series of conversion of text to keyboard scan codes followed by conversion to hexadecimal code was followed to convert binary code to nucleotides with a designed nucleotide mapping four bits per two base. Multiple DNA oligonucleotides sequences carrying duplicated information was termed as cassettes and each cassettes were inserted redundantly into multiple loci of the Bacillus subtilis genome. Data can be retrieved by multiple sequence alignment of the bit data sequences followed by genome sequencing without the need of template DNA or parity checks. Main drawback for the alignment based encoding was the size limit of the cassette oligonucleotides used to encode message. If it exceeds certain length, it will occur by chance in host genome. Also complete genome sequencing was needed to retrieve the data. All the models described, encoded text data in DNA, but in 2009 Ailenberg et al., BIB010 proposed an improved Huffman coding with unique primer design, for the first time, they encoded text, images and musical characters in DNA. It employed modified base 3 Huffman code by dividing the keyboard characters into three groups of DNA codon and assigning DNA bases according the frequency of their occurrence. DNA sequences was synthesised and inserted in plasmid and decoding was done by sequencing of plasmid. Initially index plasmid which consist of information like title, authors, plasmid number and primers assignments is constructed. They used specific primers with unique prefix code for different types of file, for instance, text data was initiated by "tx" and music data was initiated by "mu". Data was inserted in plasmid with unique sequencing primer for information retrieval. They used nucleotides efficiently by encoding 4.9 bases per characters and encoded 1688 bits data. 2.1.4. Church and Goldman encoding model. Although aforementioned work were cornerstone for storing data to DNA they were successful on a small scale as it encoded small bits of data. The most rewarding work was done in recent times by  , at Harvard University . Using next generation synthesis and sequencing technology, Church came up with efficient one bit per base algorithm of encoding information bits into fix length of DNA chunks (99 bases). Flanking primers at the beginning and end of information data was inserted to identify the specific DNA segment in which the particular data was encoded  . They encoded entire book (Regenesis: How Synthetic Biology Will Reinvent Nature and Ourselves ISBN-13:978-0465021758), including 53,426 words, 11 JPG images and one JavaScript program into 54,898 oligos each 159 nucleotide (nt) in length and consisting of a 96-bit data block (96 nt), a 19-bit address (19 nt) specifying the data block location and flanking 22 nt common sequences to facilitate amplification and sequencing. Initially the book to be encoded was converted into HTML format including all the images in it. The individual bits was converted to DNA sequence with conversion principle 1 bit per base encoding, A or C for 0 and T or G for 1. The bases were selected randomly avoiding homo polymer greater than 3 and constant GC content. The bits were indexed by 19 bits long bar-code sequence of consecutive number starting from 0000000000000000001 which determines the location of encoded bits within the book. Each DNA segment was of length 12 without bar-code and the total number of oligonucleotides generated was 5.27 MB. Specific primer sequence of 22 nucleotide for the sequencing was designed and amplified using PCR. The sequence was read using an Illumina HiSeq next generation sequencer. In writing and reading DNA, 10 bits error occurred from 5.27 MB. It has only one drawback of lacking error correction scheme that was taken care by Goldman with 100 percent of data retrieval. In 2013 Goldman used one bit per base system introduced by Church and modifying it by employing the improved base 3 Huffman coding (trits 0, 1 and 2). In this original file in binary code (0, 1) is converted to a ternary code (0, 1, 2), which is in turn converted to the triplet DNA code. It involved four steps shown in Fig 5. Binary Stepwise encoding of data into DNA using Goldman's approach is elucidated in detail. Binary data converted to base 3 Huffman code which then converted to DNA sequences. Each DNA sequences converted to fragments with each 75 base pairs overlapped in alternate fragment with reverse complement. digits holding the ASCII codes was converted to base-3 Huffman code that replaces each byte with five or six base-3 digits (trits). Each of trit was encoded with one of the three nucleotides different from the previous one used to avoid homo polymers that cause error in synthesis of DNA. DNA strand was divided into chunks each of length 117 base pair (bp). 75 bases for each DNA information chunks were overlapped with four fold redundancy to recover the data loss that occurred during synthesis and sequencing DNA. For the data security each redundant chunk was converted to reverse complement of the strand in every alternate chunks. Each DNA chunk was appended with data address blocks of 117 bases to determine the location of segment in overall data. One parity check bit was added for the intra file location and error detection. Total 153,335 DNA strings were generated. 33 nucleotide base primer was added to facilitate synthesis process and amplification. For details reader is referred to BIB012 . As proof of concept, they used four different file types (739 kilobytes file size) and achieved 2.2 PB/g DNA storage density.