LAYERED CODING ARCHITECTURES FOR NUCLEIC ACID MEMORY
Described herein are approaches allowing the storing of data at lower densities and increased write speeds. Indexing and recording of data may be separated into separate processes. Rapid DNA extension reactions can then be performed at many distinct locations throughout a solid support, so that the write speed is limited by the ability of the instrumentation to perform spatial addressing operations, rather than chemical synthesis steps.
1 . A method of storing data in a nucleic acid sequence wherein bits in a dataset are defined by a combination comprising:
an index sequence,
a layer number; and
a presence of a specific nucleotide or nucleotide analog.
2 . The method of claim 1 wherein the combination specifying bits in the dataset further comprises an absence of a specific nucleotide or nucleotide analog.
3 . The method of claim 1 wherein each of a plurality of nucleic acids at a discrete physical location comprise the same index sequence.
4 . The method of claim 3 wherein the recording media comprises a plurality of discrete locations and the plurality of nucleic acids at each of the plurality of discrete locations comprise a unique index sequence.
5 . The method of claim 1 wherein the layer number in a DNA sequence is specified by a number of layer boundaries between a region of interest and the index region.
6 . The method of claim 5 wherein at least one specific nucleotide is used to indicate a layer boundary in a sequence.
7 . The method of claim 2 wherein the presence or absence of each nucleotide or nucleotide analog corresponds to a write cycle.
8 . The method of claim 7 wherein each write cycle in each layer corresponds to a distinct nucleotide or nucleotide analog.
9 . A system for storing data in nucleic acid sequences, the system comprising:
media comprising a solid support, wherein the solid support comprises a plurality of regions of covalently linked DNA strands, and wherein the covalently linked DNA strands in each of the plurality of regions comprises a unique index sequence.
10 . The system of claim 9 further comprising a spatially addressable head operable to deliver one or more of nucleic acid synthesis reagents or decaging stimulus to each of the plurality of regions individually.
11 . The system of claim 9 wherein the plurality of regions is in excess of 100 million
12 . The system of claim 9 wherein the plurality of regions is in excess of 1 billion
13 . The system of claim 9 wherein the plurality of regions is in excess of 10 billion
14 . The system of claim 9 wherein the plurality of regions is in excess of 100 billion
15 . The system of claim 9 wherein the unique index sequence is generated using in situ synthesis
16 . The system of claim 15 wherein the in situ synthesis is performed with a template-independent polymerase from an initiator sequence.
17 . The system of claim 9 wherein the unique index sequence is immobilized through mechanical deposition of pre-synthesized sequences onto a surface
18 . The system of claim 9 wherein the unique index sequence is randomly deposited from a complex library and amplified to higher densities
19 . The system of claim 18 wherein the complex library is synthesized using a template independent polymerase and a mixture of trisphosphates
20 . The system of claim 18 wherein the complex library comprises sequence fragments that are isolated from biological sources
21 . A method for copying DNA recording media, the method comprising:
preparing a DNA-indexed template wafer comprising DNA-patterned sites separated by hydrophobic regions wherein DNA strands in the DNA-patterned sites comprise an index element flanked by a different primer binding region on both its 5′ and 3′ sides;
preparing a blank wafer comprising DNA-patterned sites separated by hydrophobic regions wherein DNA strands in the DNA-patterned sites contain the same 5′-primer binding site as the DNA-indexed template wafer;
forming droplets at each DNA-patterned site on the DNA-indexed template wafer;
aligning the DNA-patterned sites of the DNA-indexed template wafer with the DNA-patterned sites of the blank wafer such that the droplets form water columns bridging the DNA-patterned sites of the DNA-indexed template wafer and blank wafer;
annealing a primer to the 3′-primer binding site on the DNA-indexed template wafer;
performing template-dependent extension of the primer to copy the DNA strands in the DNA-patterned sites of the DNA-indexed template wafer;
denaturing the copied sequence;
re-annealing at least a portion of the copied sequence to the DNA strands in the DNA-patterned sites of the blank wafer;
performing template-dependent extension on the re-annealed portions of the copied sequence;
repeating the process of denaturation, re-annealing, and template-dependent enzymatic extension steps until a desired oligonucleotide density is achieved on the blank wafer.
22 . A method for recording data to a DNA-patterned recording media using spatially selective chemical reactions wherein the occurrence or non-occurrence of a spatially selective chemical reaction is used to encode data as a binary bit ‘1’ or ‘0’
23 . The method of claim 22 wherein an extent of the spatially selective chemical reaction encodes data using bit values above base-2 encoding
24 . The method of claim 22 wherein the spatially selective chemical reactions are local electrochemical reactions conducted with a microelectrode array
25 . The method of claim 22 wherein the spatially selective chemical reactions are mechanical deliveries of a buffered reaction mixture comprising a template independent polymerase and a deoxyribonucleotide triphosphate
26 . The method of claim 22 wherein the spatially selective chemical reactions are localized photolysis
27 . The method of claim 26 wherein localization of the photolysis reactions is controlled with a spatial light modulator
28 . The method of claim 27 wherein the photolysis is conducted with electromagnetic radiation of a wavelength between 300-410 nm
29 . The method of claim 27 wherein the photolysis reaction is conducted with electromagnetic radiation of a wavelength between 450 and 700 nm
30 . The method of claim 27 , wherein the spatially modulated light is generated from the interference pattern of at least two coherent light sources
31 . The method of claim 22 , wherein the occurrence of a spatially selective chemical reaction facilitates the template-independent enzymatic extension of at least a fraction of oligonucleotides comprising a reaction site with a dNTP
32 . The method of claim 31 , wherein the spatially selective chemical reaction removes a protecting group from the terminal nucleotide of a surface bound initiator which otherwise prevents extension of that initiator with a template-independent polymerase.
33 . The method of claim 31 , wherein the spatially selective chemical reaction removes a protecting group from a dNTP which otherwise prevents its polymerization with a template-independent polymerase.
34 . The method of claim 31 , wherein a plurality of write steps are conducted by utilizing a defined series of dNTPs so that each distinguishable dNTP corresponds to a cycle of data writing.
35 . The method of claim 34 , wherein the defined series of dNTPs are reused after addition of a distinct intervening region of sequence, the distinct intervening region of sequence serving as a layer boundary and separating layers of encoded data.
36 . The method of claim 34 , wherein the plurality of write steps are conducted on a DNA-patterned recording media prior to appending unique indices to each location
37 . The composition of claim 36 , wherein the recording media comprises a solid support patterned with at least one common initiator sequence at every desired synthesis location
38 . The method of claim 37 , wherein the unique indices are enzymatically synthesized in situ using data encoding strands as initiators after the data has been recorded
39 . A method for recovering DNA-encoded data, the method comprising:
sequencing data-encoding DNA strands;
grouping sequencing reads by index sequences;
aligning the grouped sequence reads by layers;
translating an occurrence, extent, or absence of a writing reaction at each write cycle, in each layer, for each indexed group into a bit of data;
combining bits of data from each write cycle, in each layer, for each indexed group to create a dataset.
40 . The method of claim 39 further comprising separating the data-encoding DNA strands from a recording media before sequencing.
41 . The method of claim 40 further comprising selecting depth for the sequencing step to compensate for partial addition reactions during data writing operations.
42 . The method of claim 40 further comprising determining the layers by identifying layer boundary nucleotides.
43 . The method of claim 40 further comprising altering sequencing depth by selecting for specific size ranges of the data-encoding DNA strands.