DESCRIPTION

STOICHIOMETRIC NUCLEIC ACID PURIFICATION USING RANDOMER

CAPTURE PROBE LIBRARIES

This application claims the benefit of United States Provisional Patent Application No 62/332,778, filed May 6, 2016, the entirety of which is incorporated herein by reference.

BACKGROUND

Technologies for writing (Gene Synthesis), editing (CRISPR CAS) and reading (Next generation Sequencing-NGS) large collections of nucleic acids requires an enormous (>1000) number of oligonucleotides to be used as building blocks (writing), guides (editing) or hybridization probes and primers for doing highly multiplexed enrichment and sequencing (reading). It is not economical to synthesize, purify, and quantitate each oligonucleotide individually. Companies such as Agilent, NimbleGen, and Twist Biosciences have developed array-based synthesis platforms to allow highly multiplex DNA oligonucleotide synthesis, but the oligonucleotides synthesized by these platforms include truncation products. Because modern oligonucleotide synthesis occurs from 3' to 5', most impurity species are truncation oligonucleotide products lacking a number of nucleotides at the 5' end, followed by species with one or more internal deletions. In single-plex synthesis, these fraction of these impurity products can be reduced through post-synthesis high pressure liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) purification, but HPLC and PAGE cannot be used to purify a pool of many different oligonucleotides. Furthermore, HPLC and PAGE are time- and labor-intensive and cannot be easily automated to high throughput. Additionally, even single-plex HPLC and PAGE purification of oligonucleotides result only in below 90% purity of full-length oligonucleotide products.

The concentrations of different oligonucleotides in an array- synthesized pool will vary significantly based on oligonucleotide length, oligonucleotide sequence, and synthesis reagent age and purity. Consequently, oligonucleotides synthesis yields can vary by more than 16-fold from the same quantity of initial synthesis reagents. The variation in oligonucleotide concentrations can adversely affect downstream applications, e.g. , in the production of long synthetic genes. In NGS, concentration variations in oligonucleotide pools used for hybrid-capture enrichment result in sequencing biases that cause significant wasted NGS reads. SUMMARY

In accordance with the present disclosure, there is provided a method for generating a set of precursor nucleotide sequences comprising a target oligonucleotide molecule, wherein the precursor nucleotide sequence comprises a fifth region that comprises the nucleotide sequence of the target oligonucleotide molecule and a fourth region and a third region, wherein at least one of the fourth and third regions differs from any subsequence within the target oligonucleotide, the method comprising:

(i) calculating for the precursor nucleotide sequence the standard free energy of hybridization between the precursor nucleotide sequence and (a) a first oligonucleotide comprising a second region that is complementary to the third region of the precursor oligonucleotide sequence, and (b) a first region that is complementary to the fourth region of the precursor oligonucleotide sequence;

(ii) calculating the standard free energy of a capture reaction as the standard free energy of hybridization between the precursor nucleotide sequence and the first oligonucleotide and the standard free energy of hybridization between the first oligonucleotide and target oligonucleotide;

(iii) rejecting the precursor nucleotide sequence if the standard free energy of the capture reaction does not meet a certain criterion; and

(iv) repeating steps (i) to (iii) until a set of precursor nucleotide sequences meets the criteria; and

(v) producing the set of precursor nucleotide sequences.

The criteria may be a negative free energy;

In another embodiment, there is provided a method for producing a set pf precursor nucleotide sequences comprising a plurality of barcode sequences, comprising:

(i) generating a set of precursor nucleotide sequences comprising each target oligonucleotide molecule, wherein each precursor nucleotide sequence comprises a third region that is conserved across all precursor nucleotide sequences, a fourth region that is unique for each target oligonucleotide molecule, and an fifth region that comprises the nucleotide sequence of a target oligonucleotide molecule;

(ii) calculating for each precursor nucleotide sequence the standard free energy of hybridization between the precursor nucleotide sequence and (a) a first oligonucleotide, comprising a second region that is complementary to the third region of the precursor oligonucleotide sequence, and (b) a first region that is complementary to the fourth region of the precursor oligonucleotide sequence;

(iii) calculating for each precursor nucleotide sequence the standard free energy of hybridization of folding;

(iv) calculating the standard free energy of a capture reaction as the standard free energy of hybridization between the precursor nucleotide sequence and the first oligonucleotide and the standard free energy of folding of the first oligonucleotide and the standard free energy of folding of the precursor oligonucleotide;

(v) rejecting the set of precursor nucleotide sequences if the standard free energy of the capture reaction for any precursor nucleotide sequence exceeds a certain criteria;

(vi) repeating steps (i) to (v) until a set of precursor nucleotide sequences meets the criteria; and

(vii) producing said set of precursor nucleotide sequences.

The criteria may be a selected from the group consisting of a maximum range of standard free energies of capture, a standard deviation of standard free energies of capture, and a difference between two ranks in a sorted list. The criteria may be a maximum range of no more than 5 kcal/mol between a lowest standard free energy of capture and a highest standard free energy of capture for the set of precursor nucleotide sequences. The maximum range may be no more than 2 kcal/mol.

In yet another embodiment, there is provided a method for purifying one or multiple target nucleic acid molecules from a sample comprising one or a plurality of species of precursor molecules, wherein each species of precursor molecule comprises an fifth region comprising a target nucleic acid molecule sequence, a fourth region comprising a sequence unique to the species of precursor molecule in the plurality of species of precursor molecules, defined as a barcode sequence of length n, wherein 2" is greater than or equal to the number of unique target nucleic acid molecule sequences, a third region that is conserved across all precursor molecules, the method comprising:

contacting the sample with a capture probe library at temperature and buffer conditions conducive to hybridization;

wherein the capture probe library comprises a plurality of capture probe species, wherein each capture probe species comprises a first oligonucleotide comprising a first region comprising a nucleotide sequence of n nucleotides in length and a second region that is conserved across all capture probe species, wherein each nucleotide in the nucleotide sequence of n nucleotides in length is selected from two or more nucleotides and the first region is unique to each capture probe, and wherein the second region is complementary to the third region, and wherein the fourth region of each species of precursor molecule is complementary to the first region of a species of precursor molecule;

separating the plurality of species of precursor molecules hybridized to the plurality of capture probe species from the species of precursor molecules not hybridized to the plurality of capture probe species;

treating the plurality of species of precursor molecules hybridized to the plurality of capture probe species with a cleavage agent sufficient to site-specifically cleave the plurality;

of species of precursor molecules at a site to separate the fifth regions from at least a portion of the third and fourth regions;

recovering the fifth regions from the plurality of capture probe species and the at least a portion of the third and fourth regions, and

thereby producing a purified target nucleic acid molecule or molecules.

Each capture probe species may further comprise a second oligonucleotide comprising a ninth region, wherein the ninth region is complementary to the second region. Each first oligonucleotide may further comprise a seventh region, each second oligonucleotide further comprises an eight region, and wherein the seventh region is complementary to the eight region. Each first oligonucleotide further may comprise a chemical moiety, and wherein the separating the plurality of species of precursor molecules hybridized to the plurality of capture probe species comprises surface capture of the chemical moiety. The chemical moiety may be selected from the group consisting of biotin, a thiol, an azide, an alkyne, a primary amine and a lipid. The first nucleotide hybridized with the precursor oligonucleotide may be the preferred ligand of an antibody or other receptor that mediate the surface capture of the complexes.

Recovering the fifth regions from the plurality of capture probe species and the at least a portion of the third and fourth regions may comprise a treatment selected from the group consisting of heating, introducing denaturants, washing with low salinity buffers, and introducing a nuclease. The site-specific cleavage may comprise a treatment selected from the group consisting of changing the temperature, changing the pH, and illuminating the plurality of species of precursor molecules hybridized to the plurality of capture probe species at a specific wavelength. The standard free energies of binding between each first oligonucleotide and a DNA sequence complementary to the entire sequence of the first oligonucleotide may be within 5 kcal/mol of each other. The two or more nucleotides at each nucleotide in the nucleotide sequence of n nucleotides in length maybe A or T, or may be G or C. The two or more nucleotides at each nucleotide in the nucleotide sequence of n nucleotides in length may be G or C for one or more nucleotides in the nucleotide sequence and A or T for one more nucleotides in the nucleotide sequence. The first region may comprise between 3 and 25 nucleotides, n may be between 3 and 60, 3 and 18, or 3 and 10 and not greater than the number of nucleotides in the first region. The first region may further comprise at least one nucleotide in addition to the nucleotide sequence of n nucleotides in length. The second region may further comprise between 8 and 200 nucleotides.

The barcode sequence of each species of precursor molecule is assigned based on a method comprising:

generating a set of precursor nucleotide sequences comprising each target oligonucleotide molecule, wherein each precursor nucleotide sequence comprises a third region that is conserved across all precursor nucleotide sequences, a fourth region that is unique for each target oligonucleotide molecule, and an fifth region that comprises the nucleotide sequence of the target oligonucleotide molecule;

calculating for each precursor nucleotide sequence the standard free energy of hybridization between the precursor nucleotide sequence and a first oligonucleotide, comprising a second region that is complementary to the third region of the precursor oligonucleotide sequence, and a first region that is complementary to the fourth region of the precursor oligonucleotide sequence;

calculating for each precursor nucleotide sequence the standard free energy of hybridization of folding;

calculating the standard free energy of a capture reaction as the standard free energy of hybridization between the precursor nucleotide sequence and the first oligonucleotide and the standard free energy of folding of the first oligonucleotide and the standard free energy of folding of the precursor oligonucleotide;

rejecting the set of precursor nucleotide sequences if the standard free energy of the capture reaction for any precursor nucleotide sequence exceeds a certain criteria; and repeating the method until a set of precursor nucleotide sequences meets the criteria. In still yet another embodiment, there is provided a capture probe library comprising: a plurality of oligonucleotides comprising a first plurality of oligonucleotides wherein each oligonucleotide of the first plurality of oligonucleotides comprises:

a first region comprising a first nucleotide sequence comprising at least 3 variable positions, wherein each variable position comprises a nucleotide selected from at least two possible nucleotides,

wherein the first nucleotide sequence comprising at least 3 variable positions is unique to each oligonucleotide, and

a second region comprising a second nucleotide sequence, wherein the second nucleotide sequence of the second region is conserved across each oligonucleotide in the first plurality of oligonucleotides,

A capture probe library comprising:

a plurality of oligonucleotides comprising a first plurality of oligonucleotides and a second plurality of oligonucleotides, wherein each oligonucleotide in the first plurality of species of oligonucleotides comprises

a first region comprising a nucleotide sequence comprising at least 3 variable positions, wherein each variable position comprises a nucleotide selected from at least two possible nucleotides,

wherein the nucleotide sequence comprising at least 3 variable positions is unique to each species of oligonucleotide, and

a second region comprising a nucleotide sequence, wherein the nucleotide sequence of the second region is conserved across each oligonucleotide in the first plurality of oligonucleotides,

wherein each oligonucleotide in the second plurality of oligonucleotides comprises a third region, wherein the third region is complementary to the second region.

The standard free energies of binding between each oligonucleotide in the first plurality of oligonucleotides and a DNA sequence complementary to the entire sequence of the respective oligonucleotide in the first plurality of oligonucleotides may be within 5 kcal/mol of each other. The at least two possible nucleotides at each nucleotide in may be A or T, or may be G or C. The

at least two possible nucleotides at each nucleotide in may be G or C for one or more nucleotides in the nucleotide sequence and A or T for one more nucleotides in the nucleotide sequence. The concentration of a second oligonucleotide may be greater than a sum of the concentrations of each oligonucleotide of the first plurality of oligonucleotides. The first region may comprise between 3 and 25 nucleotides. The number of variable positions in the first region may be between 3 and 60, between 3 and 18, or between 3 and 10, and not greater than a total number of nucleotides in the first region. The first region further may comprise at least one nucleotide in addition to the at least 3 variable positions. The second region may comprise between 8 and 200 nucleotides. The at least three variable regions may be contiguous or non-contiguous.

A further embodiment comprise an oligonucleotide library for the multiplexed capture of a set of desired precursor nucleic acid molecules comprising:

a plurality of species of precursor molecules, wherein each species of precursor molecule comprises

a third region comprising a nucleotide sequence that is conserved across all species of precursor molecules,

a fourth region comprising a barcode sequence comprising 3-60 nucleotides, an fifth region comprising a target nucleic acid molecule sequence that is unique to the species of precursor molecule in the plurality of species of precursor molecules, wherein the barcode sequence of each species of precursor molecule is different and wherein 2" is greater than or equal to the number of unique target nucleic acid molecule sequences; and

a capture probe library comprising a plurality of capture probe species, wherein each capture probe species comprises an oligonucleotide comprising a first region comprising a nucleotide sequence of n nucleotides in length, a second region that is conserved across all capture probe species, wherein each nucleotide in the nucleotide sequence of n nucleotides in length is selected from two or more nucleotides and the first region is unique to each capture probe, and wherein the second region is complementary to the third region, and wherein the fourth region of each species of precursor molecule is complementary to the first region of a species of precursor molecule.

The the standard free energies of binding between each species of first oligonucleotide in the plurality of capture probe species and a DNA sequence complementary to the entire sequence of the respective species of first oligonucleotide may be within 5 kcal/mol of each other. The two or more nucleotides at each nucleotide in the nucleotide sequence of n nucleotides in length may be A or T, or may be G or C. The two or more nucleotides at each nucleotide in the nucleotide sequence of n nucleotides in length may be G or C for one or more nucleotides in the nucleotide sequence and A or T for one more nucleotides in the nucleotide sequence. The a concentration of a second oligonucleotide may be greater than a sum of the concentrations of each species of first oligonucleotide. The first region may comprise between 3 and 25 nucleotides, n may be between 3 and 60, 3 and 18, or 3 and 10, and not greater than the number of nucleotides in the first region. The first region may further comprise at least one nucleotide in addition to the nucleotide sequence of n nucleotides in length. The second region may comprise between 8 and 200 nucleotides. The barcode sequence of each species of precursor molecule may be selected based on the method as set forth above. The at least one species of precursor molecule may be chemically synthesized or produced enzymatically. The enzyme used to produce the at least one species of precursor molecule may be a ligase or a polymerase.

In an additional embodiment, there is provided a method for purifying multiple target nucleic acid molecules from a sample comprising a plurality of precursor molecules, wherein the method comprises the steps of:

providing a plurality of nucleic acid probes, wherein each probe has a sequence complementary to a region of one of the precursor molecules and a first moiety sufficient to allow isolation of the probe;

adding the plurality of nucleic acid probes to a sample comprising the plurality of precursor molecules under conditions sufficient to promote hybridization of each nucleic acid probe to the region of the precursor molecule complimentary to the sequence thereby forming a plurality of probe- precursor complexes, wherein each precursor molecule comprises a different target nucleic acid molecule, and wherein the region of each precursor molecule does not comprise the target nucleic acid molecule; and

isolating the plurality of probe-precursor complexes via interaction with the first separating the target nucleic acid molecules from the plurality of probe-precursor.

Another embodiment provides for a method for producing a plurality of distinct target oligonucleotides each having a specified sequence, the method comprising:

(1) synthesizing a precursor oligonucleotide for each distinct target oligonucleotide, wherein the precursor comprises a third sequence, a fourth sequence, and a fifth sequence, wherein the third sequence is identical for all precursors, the fourth sequence comprises a barcode and is distinct for all precursors, and a fifth sequence corresponds to the target sequence,

(2) synthesizing a capture probe library of distinct oligonucleotides comprising a first sequence and a second sequence, wherein the first sequence comprises degenerate randomer nucleotides and wherein at least one first sequence is complementary to each fourth sequence, and the second sequence is complementary to the third sequence,

(3) mixing the precursors and the capture probe library in an aqueous hybridization buffer,

(4) removing precursor molecules not bound to the capture probes,

(5) enzymatically or chemically cleaving the fifth sequence from the remainder of the precursor molecules, and

(6) removing the capture probe library and the remainder of the precursor molecules.

The first sequence may comprise an S degenerate nucleotide at certain positions and/or a W degenerate nucleotide at certain positions, but may not comprise an N degenerate nucleotide at any position, such that any degenerate variant of the first sequence is complementary to one or more fourth sequences, wherein S is a strong base, W is a weak base, and N is any base. The capture probes may be functionalized with a moiety permitting for rapid binding to a ligand, such as a moiety selected from a biotin, a thiol, an azide, or an alkyne. In step (4), eliminating precursor molecules not bound to capture probes may comprise adding particles that specifically bind the capture probe, followed by removal of supernatant solution. The particles may be selected from streptavidin-coated magnetic beads or streptavidin-coated agarose beads. The precursors may further comprise a deoxyuracil nucleotide or an RNA nucleotide, and cleavage of the fifth sequence comprises introduction of a uracil DNA glycosylase or an RNAse enzyme. The precursors may further comprise a photolabile or heat-labile moiety, and the cleaving of the fifth sequence comprises exposure of the solution to light of the appropriate wavelength or heating to the appropriate temperature. The fourth sequence may further comprise the "S" degenerate nucleotide at certain positions and/or the "W" degenerate nucleotide at certain positions, but does not comprise the "N" degenerate nucleotide at any position. The length of the first sequence may be between 5 and 50 nucleotides, and wherein the number of degenerate nucleotides is between 1 and 30, between 1 and 20, between 1 and 10, between 2 and 8, between 2 and 6, or between 3 and 5. The length of the second sequence may be between 5 and 50 nucleotides, and/or wherein the length of each target oligonucleotide is between 5 and 500 nucleotides.

In an additional embodiment, there is provided an oligonucleotide capture probe library comprising a first sequence and a second sequence, wherein the first sequence comprises degenerate randomer nucleotides comprising an "S" degenerate nucleotide at one or more positions and/or a "W" degenerate nucleotide at one or more positions, but does not comprise an "N" degenerate nucleotide at any position, and wherein the length of the first sequence is between 5 and 50 nucleotides, the number of degenerate nucleotides is between 1 and 30, and the length of the second sequence is between 5 and 50 nucleotides. The second sequence may comprise an "S" degenerate nucleotide at certain positions and/or a "W" degenerate nucleotide at certain positions, but does not comprise a "N" degenerate nucleotide at any position. The oligonucleotide capture probe library may be functionalized with a chemical moiety for rapid binding, selected from a biotin, a thiol, an azide, or an alkyne. The one or more of the oligonucleotide capture probes may further compris a deoxyuracil nucleotide or an RNA nucleotide. The one or more of the oligonucleotide capture probes may further compris a photolabile or heat-labile moiety. The length of the first sequence may be between 5 and 50 nucleotides, and wherein the number of degenerate nucleotides may be between 1 and 30. The length of the second sequence may be between 5 and 50 nucleotides. The library may have at least 8, at least 32, or at least 256 members. The library may have between 8 and 32 members, between 8 and 256 members, between 32 and 256 members, between 8 and 1024 members, between 32 and 1024 members, or between 256 and 1024 members. The library may be found on one or more substrates.

In still an additional embodiment, there is provided an aqueous solution comprising an oligonucleotide capture probe library, a plurality of precursor oligonucleotides and a set of precursor oligonucleotides, wherein:

the capture probe library comprises a first sequence and a second sequence, wherein the first sequence comprises degenerate randomer nucleotides comprising an "S" degenerate nucleotide at certain positions and/or an "W" degenerate nucleotide at certain positions, but does not comprise an "N" degenerate nucleotide at any position, and wherein the length of the first sequence is between 5 and 50 nucleotides, the number of degenerate nucleotides is between 1 and 30, and the length of the second sequence is between 5 and 50 nucleotides,

each of the plurality of precursor oligonucleotides comprises a third sequence, a fourth sequence, and a fifth sequence, wherein the third sequence is identical for all precursors, the fourth sequence comprises a barcode and is distinct for all precursors, the second sequence is complementary to the third sequence, and at least one instance of a first sequence is complementary to each fourth sequence. The second sequence of the oligonucleotide capture probe library may comprise an "S" degenerate nucleotide at certain positions and/or a "W" degenerate nucleotide at certain positions, but may not comprise a "N" degenerate nucleotide at any position. The oligonucleotide capture probe library may be functionalized with a chemical moiety for rapid binding, selected from a biotin, a thiol, an azide, or an alkyne. The one or more of the oligonucleotide capture probes further may comprise a deoxyuracil nucleotide or an RNA nucleotide. The one or more of the oligonucleotide capture probes may further comprise a photolabile or heat-labile moiety. The length of the first sequence of the oligonucleotide capture probe library may be between 5 and 50 nucleotides, and the number of degenerate nucleotides may be between 1 and 30. The length of the second sequence of the oligonucleotide capture probe library may be between 5 and 50 nucleotides. The oligonucleotide capture probe library may have at least 8, at least 32, or at least 256 members. The oligonucleotide capture probe library may have between 8 and 32 members, between 8 and 256 members, between 32 and 256 members, between 8 and 1024 members, between 32 and 1024 members, or between 256 and 1024 members. The oligonucleotide capture probe library may be found on one or more substrates. The precursors may further comprise a deoxyuracil nucleotide or an RNA nucleotide, and cleavage of the fifth sequence comprises introduction of a uracil DNA glycosylase or an RNAse enzyme.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The word "about" means plus or minus 5% of the stated number.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Fig. 1: Schematic overview of one embodiment of Stoichiometric Nucleic Acid Purification (SNAP). A sample mixture containing two precursors of oligonucleotide targets also contains a number of truncation synthesis products that are not desired. Through the course of the method described in this disclosure, full length target oligonucleotides are produced in roughly equal stoichiometry.

Fig. 2: Schematic of a capture probe library and corresponding set of target sequences. Within the targets libraries, region 3 is conserved while regions 4 and 5 are different for each target sequence. Similarly, within a library of capture probes region 2 is conserved, while region 1 contains variable positions with two or more nucleosides being possibly present at each variable position. (SEQ ID NOS: 404-409)

Fig. 3: Multiplexed oligonucleotide purification workflow. The process consists of 6 sequential steps. In step 3, solid phase consists of functionalized magnetic beads that enable the phase separation in steps 4 and 6. Depending on whether the user desires target sequences with or without the universal region 3 and barcode region 4, in step 5 either heat/NaOH denaturation or restriction enzyme cleavage can be used after the purification to separate the final products from the library and magnetic beads.

Fig. 4: Removal of regions 3 and 4 from target sequences using an enzyme. The desired region 5 can be enzymatically cleaved from the 5' regions 3 and 4 used for purification, for applications where such sequences are undesirable. Site-specific cleavage can be implemented through use of (a) uracil DNA glycosylase alone or in a formulation containing DNA glycosylase-lyase Endonuclease VIII - commercially known as USER, (b) RNAse, (c) Fokl endonuclease, or other methods known to one of ordinary skill in the art. The sequence or chemical composition of regions 3 and 4 may be adapted to accommodate the anticipated enzymatic cleavage process. (SEQ ID NOS: 410-417 and 251) Fig. 5: Use of multiple barcodes for the same target sequence increases its concentration in the final purified mixture. Degenerate nucleotides (i. e. , S = G or C and W = A or T) within the barcode region 4 facilitates the achievement of arbitrary desired stoichiometric ratios of targets by avoiding individual synthesis of a number numbers of precursor oligonucleotides. (SEQ ID NOS: 418-421)

Fig. 6: Proof of concept results using fluorescent labelled oligonucleotides, assayed through fluorescent polyacrylamide gel electrophoresis. 160 pmol of a capture probe library biotinylated in 3 'end, comprising 64 different region 1 sequences, is incubated with 64 synthetic unpurified precursor oligonucleotides each at 10 pmol. Each precursor oligonucleotide has a uniquely assigned region 4, complementary to one and only one instance of region 1 on the capture probe species. Lane 1 shows the length distribution of Precursor 1, and Lane 2 shows the length distribution of Precursor 1 after undergoing SNAP, but without cleavage of regions 3 and 4. Lane 3 shows the SNAP product after cleavage of regions 3 and 4. Lane 4 shows the length distribution of the corresponding commercially provided PAGE-purified oligo as comparison. Lanes 5-10 show that, regardless of initial stoichiometric ratio of the 3 Precursors, the final stoichiometry of the SNAP products are close to 1: 1: 1. The SNAP protocol used for this series of experiments is as follows: The capture probe library and Precursors are allowed to hybridize for 2 hr at 60 °C in 0.5M NaCl pH 7.5. Subsequently, 3 mg of streptavidin coated magnetic beads (pre-washed with the same incubation buffer) are added to the oligonucleotide mixture to a final volume of 100 μί, and incubated for 30 min at 60 °C, with the sample being rocked by a shaker. The supernatant is then discarded and the magnetic beads are washed twice in the incubation buffer and one time in the buffer to be used for the subsequent enzymatic cleavage. For lane 2, the full-length precursors are eluted using 50% v/v formamide at 95 °C for 5 to 15 min. For lane 3, the beads were re-suspended in USER buffer, and 2 enzymatic units of USER enzyme mix were added to achieve a final volume of 25 μί; this mix was then incubated for 1 hour at 37 °C. Finally supernatant containing the desired purified target is extracted.

Figs. 7A-B. Characterization of purity and stoichiometry for multiplexed SNAP. 64 separate Precursors (10 pmol each) were simultaneously purified via SNAP. (Fig. 7 A) Next Generation Sequencing was used to characterize the purity of the 64 oligos, where the purity is operationally defined as the number of perfectly aligned reads divided by the number of reads aligned by Bowtie 2. The purity of the SNAP products are significantly higher than that of individually PAGE purified oligonucleotides (median 79% vs. 61%). (FIG. 7B) Digital droplet PCR was used to evaluate the stoichiometries of the 64 SNAP product oligonucleotides.

Fig. 8: Characterization of purity for 256-plex SNAP purified (2.5 pmol of each Precursor).

Figs. 9A-D: Four possible variations in design of the capture probe libraries.

Fig.10: Experimental results on double-stranded capture probe article shown in Fig. 9B. SNAP was performed using 640 pmol of capture probe biotinylated in 5'end, 1280 pmol of protector strand, and 40 pmol of one unpurified Precursor. Note that, unlike in Fig. 6, only a single Precursor species is introduced, so that the Capture Probes are NOT saturated by Precursors. SNAP protocol is otherwise similar to Fig. 6 caption. Lane 1 corresponds to the unpurified Precursor, Lane 2 corresponds to the PAGE-purified Target oligo, Lane 3 corresponds to the SNAP product without cleavage of regions 3 and 4, and Lane 4 corresponds to the final SNAP product. Lanes 5-10 show that, regardless of initial stoichiometric ratio of the 3 Precursors, the final stoichiometry of the SNAP products are close to 1: 1 : 1.

Fig. 11 : Purification of RNA transcripts produced from a synthetic DNA template that comprises regions 3 and 4.

Fig.12 Cumulative distribution of AG° _rX n for two different randomer sequences, each producing 64 instances. Using an SWSWSW library (flanked by CA in 5' end and C in 3' end) produces a tight thermodynamics range of 0.8 kcal/mol window. In contrast, using a TGANNN library results in a spread of more than 4 kcal/mol.

Figs.l3A-C: Algorithm for the design of probe libraries and assignment of barcodes (region 4). Fig. 14: Workflow for the characterization of purity of oligonucleotides libraries through NGS. The oligonucleotides share a universal region at the 3' end, which is used to align and enzymatically extend one short sequence used as primers. Upon the phosphorylation of the 5' end of the newly formed double stranded library, adaptors containing sequencing primers are enzymatically attached. Finally, the resulting library is amplified for 3 PCR cycles, to introduce the P7 and P5 sequences used for the cluster generation and the consequent sequencing with an Illumina instrument.

Fig. 15: Workflow for the characterization of stoichiometries within oligonucleotides libraries through digital droplet PCR (ddPCR). The oligonucleotides of the library share a common region at 3' end for the priming with a reverse primer, which is added as well as the master mix contain the enzyme and the EvaGreen dye for ddPCR reaction. The reaction mixture is distributed in 64 equal aliquots, each of which receives one forward primer specific for one oligonucleotide sequence within the library. Subsequently oil is added to every sample, which undergoes to the emulsion process. Finally, upon the PCR reaction, the fluorescent reader is used to determine the ratio between the droplets-containing amplification product and those that are empty, ratio that give a statistical quantification of the template molecule that have been amplified.

DETAILED DESCRIPTION

The goal of this disclosure is outlined shown in Fig. 1: different precursor oligonucleotides are synthesized with various yields and purities, and pooled together to form an input oligonucleotide pool. Through a process of SNAP purification, an output pool of target oligonucleotides is produced with no truncation products, and exhibiting a desired stoichiometric ratio (1 : 1 in Fig. 1).

In certain aspects of the present disclosure, toehold probes with a randomer toehold sequence are used to capture artificially designed 5' sequence of the target oligonucleotides. Because the probes are toehold probes which are selective to single nucleotide variations, even truncated synthesis products one nucleotide shorter than the full-length product will not be efficiently captures.

I. Precursor Oligonucleotides

A full-length precursor oligonucleotide comprises three regions, labeled as 3, 4, and 5 in Fig. 2. Region 3 is also referred to as the validation region, and the sequence of region 3 is conserved across all precursor species. Region 4 is also referred to as the barcode, and the sequence of region 5 is unique to each precursor species. Region 5 is referred to as the target sequence, and is the only portion of the oligonucleotide that should remain after the purification process. Across different precursor species, some region 8's may be unique, while others may be redundant. In some embodiments, the nucleotide to the 5' of region 5 is a modified nucleotide (e.g. , a deoxyuracil nucleotide, or an RNA nucleotide) that can be site- specifically cleaved.

Because DNA synthesis (both chemical and enzymatic) is imperfect, there will exist truncation products in which precursors lack one or more nucleotides at either the 5' end (chemical synthesis) or the 3' end (enzymatic synthesis). II. Capture Probe Library

Fig. 2 shows one preferred embodiment of the capture probe library used to perform SNAP purification for chemically synthesized oligonucleotides with truncations at the 5 ' end. In this embodiment, the library comprises two types of oligonucleotides: the probe oligonucleotides (comprising regions 1 and 2). In certain aspects, the probe oligonucleotides are functionalized at the 3' end with a biotin, in order to allow capture by streptavidin- functionalized magnetic beads.

The sequence of region 1 is designed and synthesized as a randomer library, in which one or several positions contain a mixture of multiple nucleotides. The complement of every precursor species' barcode (region 4) should exist as an instance of the region 1 randomer library.

The sequence of region 2 is designed to be complementary to the sequence of region 3 on precursors.

III. Sequence Design of Randomer Region 1

The variable positions and allowable nucleotides at each variable position should be designed such that the standard free energy of hybridization of each instance region 1 to its perfect complement are similar. In some embodiments, the sequence of region 1 comprises S (strong, mixture of G and C) and W (weak, mixture of A and T) degenerate nucleotides.

As one example of an undesirable sequence construction, if region 4 is designed as a 7nt NNNNNNN region, then both GCGCGCG and TATATAT members will be present. The AGo of these two members pairing with their complements at 37 °C in 1M Na+ are -13.23 kcal/mol and -4.38 kcal/mol, respectively, according to SantaLucia Jr, J., & Hicks, D. (2004). The thermodynamics of DNA structural motifs. Annu. Rev. Biophys. Biomol. Struct., 33, 415-440. This 9 kcal/mol difference can result in the GCGCGCG member capturing its target with >99.9% yield while the TATATAT member capturing its target with <0.1% yield; such a large difference in capture yield would be clearly undesirable for achieving uniform or ratiometric product quantity/concentration distributions.

For this reason, the nucleosides present at variable positions are designed to be either S or W. That is to say, some variable positions contain either an A or T nucleoside but not G or C, while other variable positions contains G or C but not A or T. Based on published literature parameters, there is only a maximum difference of 0.17 kcal/mol per base stack for SW and for WS stacks, at 37 °C in 1M Na+. IV. Sequence-Specific Capture

In those instances where the number of different probes is equal the number of the target sequences, and the total concentration of probes is lower than the total concentration of target, any instance of region 1 only hybridizes to its perfectly complement in region 4, as any other non-specific hybridization will be outcompeted.

Consequently, if the probe oligonucleotide library is synthesized such that all instance sequences are equally represented, and if the concentration of all precursors exceed that of their corresponding probe sequences, then the amount of precursor captured should be roughly stoichiometric, regardless of the initial stoichiometric ratio between the precursors. As a numerical example, if the sequence of region 1 is "GWSWSWST", then there are 2 ⁶ = 64 instance sequences. Assuming a total probe concentration of 6.4 μΜ, each sequence instance would have a concentration of approximately 100 nM. For an initial precursor pool in which the concentrations of each precursor species ranges between 200 nM and 10 μΜ, the amount of each precursor bound to the probe will be limited by probe instance sequence concentration to a maximum of 100 nM, except insofar as off-target hybridization between precursors and their non-cognate probe instance sequences hybridize.

As another mathematical example, a probe library with 12 variable position, and 2 possible nucleotides at each position is comprised of 2 ¹² = 4096 members. Assuming a total amount of 4 nanomoles (nmol) of the library, each member is expected to be present at quantity of roughly 1 pmol. This library is suitable for purification of up to 4096 targets, each with quantity of > 10 pmol. Array oligonucleotide synthesis providers often produce panels of oligonucleotides at either the 10 pmol or 100 pmol scale.

V. Separation of Precursors Bounded to Probes

The precursor oligonucleotides bound to the probe oligonucleotides are separated from other precursors using the probes as marker for recovery, through the use of a solid support or enzymatic degradation of unbound molecules, for example, using an exonuclease (e.g., , 5 '-3') for single-strand digestion. In a particular embodiment, the probe oligonucleotides are biotin-functionalized at the 3 ' end, and streptavidin-functionalized magnetic beads are added to solution after the hybridization reaction between the precursors, protectors, and probes. Washing the magnetic bead suspension in the vicinity of a magnetic removes unbound molecules. VI. Removal of Regions 3 and 4 from Targets

For many applications with purified pools of target oligonucleotides, the sequences of regions 3 and 4 would be an undesirable artifact. The sequence or composition of these regions may be designed to facilitate enzymatic removal of these regions from the desired target sequence after surface-based purification. Fig. 4 shows several strategies for enzymatically cleaving the captured target sequences after region 4 to remove all artifact sequences, or after region 3 to remove the purification sequence but not the barcode.

VII. SNAP Purification Workflow

Fig. 3 shows one embodiment of the overall SNAP purification workflow. Hybridization durations, buffers, and temperatures vary depending on the concentration of the probe and precursor oligonucleotides, and reasonable parameter values are known to those ordinary in the art of nucleic acid hybridization probes. The capture of biotin- functionalized probes by magnetic beads and subsequent wash protocols are typically provided by suppliers of biotin-functionalized magnetic beads (e.g. , Thermo Fisher Dynabeads, New England Biolabs streptavidin magnetic beads). The USER enzyme (e.g. , from New England Biolabs) can be used to site-specifically cleave precursor oligonucleotides at dU positions.

VIII. Ratiometric Concentrations of Purified Targets

Through the use of multiple barcodes (region 4) for the same target sequence (region 5), it is possible to adjust the stoichiometric ratios of different target sequences after SNAP purification. Fig. 5 shows an example in which 3 different target sequences are sought to be purified in a 1 :2:6 ratio.

The number of available barcodes based on variable positions determines the range of available stoichiometric ratios and number of sequences possible. For example, a probe library with 12 variable positions and 2 possible nucleotides at each position contains 2 ¹² = 4096 sequence instances. The sum of all integer stoichiometry ratios among different target sequences must sum to 4096 (or less). For example, it is possible to purify a library of 2097 target sequences, in which 2096 target sequences are at equal stoichiometry to one another, and the last target sequence is present at lOOOx excess. Importantly, degenerate randomer sequences can also be incorporated in region 4 of the precursor sequences, in order to reduce the cost of precursor synthesis. For example, in Fig. 5, Target 2 occupies two sequence instances of the barcode through the use of a degenerate W in region 7. In some instances, to yield uniform concentrations of target oligonucleotides in the final pool, the capture probe library should be at a significantly lower concentration than the input target oligonucleotide sample. For example, the full-length product of Target 1 is initially at 5 μΜ and the full-length product of Target 2 is initially at 8 μΜ, each member of the capture probe library should be kept below 5 μΜ, such as 1 μΜ. In such an instance, the purification yield may be lower than for HPLC and PAGE methods for single targets but will provide a uniform final concentration of target molecules. In instances where a uniform final target concentration is not needed, the yield will not be reduced in such a way.

IX. Barcode Assignment Based on Target Sequence

To simultaneously achieve high sequence specificity and high hybridization yield, the standard free energies of hybridization (AG°Hyb) between the different precursor and their respective matched probe sequence instances must be similar. Naive design of the validation region sequence (region 3) and assignment of barcodes (region 4) may result in precursor oligonucleotides with significant secondary structure between region 5 and regions 3 and 4, resulting in AG°Hyb significantly more positive than expected, in turn leading to lower capture yields. Consequently, it is suggested that the sequences of regions 3 and 4 be rationally designed given desired target sequences, so that similar secondary structure is observed for all precursor sequences.

X. Examples

The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 - Stoichiometric SNAP Purification

Fig. 6 shows data demonstrating proof-of-concept of the SNAP purification technique. Denaturing polyacrylamide gel electrophoresis is used to visualize and quantitate the purity and concentration of different oligonucleotide species. Lane 1 shows a chemically synthesized precursor oligonucleotide, and lane 4 shows the corresponding chemically synthesized target oligonucleotide. Both the precursor and the target oligonucleotides were synthesized with a 3' FAM fluorophore functionalization to allow easy visualization. Lanes 2 shows the captured precursor molecules before USER enzyme treatment to remove regions 3 and 4, and Lane 3 shows the final purified product after USER enzyme treatment. The relative lack of truncation bands in Lanes 2 and 3 indicate that truncation products have been removed.

Lane 5 shows a mixture of 3 precursor oligonucleotides of different lengths (lOOnt, 90nt, and 80nt), prepared at a nominal stoichiometric ratio of 1: 1:1. Lane 6 shows the output of the SNAP purification protocol. The stoichiometric ratio of the purified target oligonucleotides was quantitated to be 1.2:1: 1.5. Lane 7 and 8 show a similar set of experiments, except the 3 precursor oligonucleotides were nominally prepared at 1:5:25 and the SNAP-purified products were observed to be at 1.2:1: 1.7, and is closer to the designed 1:1:1 stoichiometric than the precursors.. Lane 9 and 10 show a similar set of experiments, except the 3 precursor oligonucleotides were nominally prepared at 5:25:1 and the SNAP- purified products were observed to be at 1.2: 1:0.5.

Figs. 7A-B show data demonstrating purity and stoichiometry attainable by means of SNAP purification as measured by Next Generation Sequence and digital droplet PCR. Fig. 7 A shows that the median of reads perfectly matching the desired sequence is about the 80%, in case of SNAP purification, which is greater that the median from PAGE purification which is about 60% and the one from unpurified oligos, which is about 55%. Fig 7B shows the concentration of the individual sequences as measured through digital droplet PCR, using target specific primers. After the SNAP purification the concentration of the oligo is within a factor of two for the 95% of the sequences of the pool.

Fig. 11 shows data demonstrating purity for a 256-plex, as measured by NGS. Also in this case the fraction of perfect reads for SNAP purified oligo is close the 80%. Example 2 -Purification of Enzymatically-produced Precursors

Fig. 10 shows an example embodiment of purifying enzymatically produced precursors, in this case a transcribed RNA species. Chemical synthesis of RNA oligonucleotides is significantly (8-fold) more expensive than DNA synthesis, and limited to shorter lengths (typically < 50nt). In vitro transcribed RNA using RNA polymerase and a corresponding DNA template sequence can produce economically produce large quantity of desired RNA target sequences that is also significantly longer than chemically synthesized RNA, but requires labor- intensive Polyacrylamide Gel electrophoresis (PAGE) purification. The present SNAP purification methods can significantly reduce the labor needed to produce RNA molecules, especially in highly multiplexed settings.

Because enzymatically produced precursors disproportionately exhibit truncations and errors at the 3' end rather than the 5 ' end, the DNA template sequence is designed so that the validation and barcode regions (3 and 4, respectively) will be positioned at the 3 ' end of the transcript. The stoichiometric capture of full-length precursor RNA transcripts occurs similarly to that of DNA oligonucleotides described previously. An RNAse H enzyme may be used to remove regions 6 and 7 from the precursor to leave only the desired target RNA sequence, because RNAse H will selectively cleave RNA at regions where it is hybridized to DNA.

Example 3 - Probe Design Variations

Figs. 9A-D show a few possible variations in design of the probe and protector sequences. The relative ordering of the regions may be altered, as long as the complementarity relationship between the regions are preserved (e.g. , region 2 is complementary to region 3).

Fig. 9B In those instances when the number capture probes is higher than the number or target sequences, as well as in those case where multiple validation regions (region 2) are used simultaneously, the purpose of the protector oligonucleotide and of regions 7 and 8, is to ensure sequence- specific hybridization of each precursor to its matched probe oligonucleotide. Zhang, D. Y., Chen, S. X., & Yin, P. (2012). Optimizing the specificity of nucleic acid hybridization. Nature chemistry, 4(3), 208-214. and Wu, L. R., Wang, J. S., Fang, J. Z., Evans, E. R., Pinto, A., Pekker, I., ... & Zhang, D. Y. (2015). Continuously tunable nucleic acid hybridization probes. Nature methods, 12(12), 1191-1196. shows that the competitive hybridization between precursors and the protectors is specific to even single- nucleotide changes in sequence when the sequences of the probe and protector are designed appropriately.

This specificity is useful for 2 purposes: First, it limits the off-target hybridization of precursors to non-cognate probe sequence instances that are not perfectly complementary. Second, it prevents the hybridization of imperfectly synthesized precursors that lack any nucleotide in regions 3 or 4.

Unless explicitly stated otherwise, "complementary" in this document refers to "partially or fully complementary". Two sequences are defined to be "partially complementary" when over 80% of the aligned nucleotides of one sequence is complementary to corresponding nucleotides of the other sequence.

Tables 1-4, below, shows a hypothetical set of sequences for the precursors, capture probe and protector that could be used in methods of the present disclosure. In the sequence of the capture probe, S or W indicates that all variants are included in the capture probe library. For example, both A and T would be present in the mixture of capture probes at each W as part of the randomer library of capture probes.

TABLE 1 - List of 64 precursor for the 64plex described in Figs. 7A-B

Name Region 3 Region 4 Region 5

NGSPIe TAGGTGC TGTGAC GCCCGTCGGCATGTATTAGCTCTAGAATTACCACAGTGCA x64_1 GGTCGCA TCACTG ACCTTTCGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 3

1 NO:2

NGSPIe TAGGTGC TGTCTG GGGGCCGGAGAGGGGCTGACCGGGTTGGTTTTGATGCG x64_2 GGTCGCA TGTCTG GTGCTCGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 5

1 NO:4

NGSPIe TAGGTGC TGTCAC CCCTGATTCCCCGTCACCCGTGGTCACCATGGTAGTGGC x64_3 GGTCGCA ACTCTG CATAGCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 7

1 NO:6

NGSPIe TAGGTGC TGTGAC TTTTTCGTCACTACCTCCCCGGGTCGGGAGTGGGTGAATT x64_4 GGTCGCA ACTCTG ATGCTGAACGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 9

1 NO:8

NGSPIe TAGGTGC TGTGAG GCCCGCCCGCTCCCAAGATCCAACTACGAGCTTTTAGGT x64_5 GGTCGCA AGACTG CAGTGGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 1 1

1 NO:10

NGSPIe TAGGTGC TGTGTC GGCCGTCCCTCTTAATCATGGCCTCAGTTCCGAAACCTAC x64_6 GGTCGCA TGTCTG ACATTATCTGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 13

1 NO:12

NGSPIe TAGGTGC TGTGAG GGTATCTGATCGTCTTCGAACCTCCGACTTTCGTTCTGGA x64_7 GGTCGCA TCACTG CATGCCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 15

1 NO:14

NGSPIe TAGGTGC TGTCTC TGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCATGCTTC x64_8 GGTCGCA AGACTG TACTCCTAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 17

1 NO:16

NGSPIe TAGGTGC TGTCAC CCTGTCCGTGTCCGGGCCGGGTGAGGTTTCCCGTGCACT x64_9 GGTCGCA TCACTG AGGGCTGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 19

1 NO:18

NGSPIe TAGGTGC TGTGAC GTAACTAGTTAGCATGCCAGAGTCTCGTTCGTTATCGGAT x64_10 GGTCGCA TGTCTG GGCCTAGTATAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 21

1 NO:20

NGSPIe TAGGTGC TGTCAC GCCCCGGACATCTAAGGGCATCACAGACCTGTTATTCCTT x64_1 1 GGTCGCA TCTCTG GTTGAAGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 23

1 NO:22

NGSPIe TAGGTGC TGTCTC GGTAGTAGCGACGGGCGGTGTGTACAAAGGGCAGGTGA x64_12 GGTCGCA AGTCTG GTATTTGATTCAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO:25

1 NO:24

NGSPIe TAGGTGC TGTCTC GGCGCTGGGCTCTTCCCTGTTCACTCGCCGTTACTATGTT x64_13 GGTCGCA TGACTG CGGCCTTTTTAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 27

1 NO:26

NGSPIe TAGGTGC TGTGTG TACCACCCGCTTTGGGCTGCATTCCCAAGCAACCCCCCC x64_14 GGTCGCA TGACTG GAAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID SEQ ID NO: 29 1 NO: 28

NGSPIe TAGGTGC TGTCTG CTTTCCCTTACGGTACTTGTTGACTATCGGTCTCGTAAAC x64_15 GGTCGCA ACACTG GGTTAGATCGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 31

1 NO: 30

NGSPIe TAGGTGC TGTCTC GGCGGACTGCGCGGACCCCACCCGTTTACCTCTTAGGTA x64_1 6 GGTCGCA TCTCTG TATAACGCCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 33

1 NO: 32

NGSPIe TAGGTGC TGTGAC GGTGGAAATGCGCCCGGCGGCGGCCGGTCGCCGGTACA x64_1 7 GGTCGCA TCTCTG CAGTTGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 35

1 NO: 34

NGSPIe TAGGTGC TGTGTG CCTTCCCCGCCGGGCCTTCCCAGCCGTCCCGGAGCAAGA x64_18 GGTCGCA ACTCTG TTGTTTACAGAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 37

1 NO: 36

NGSPIe TAGGTGC TGTCAG GGGATTCGGCGAGTGCTGCTGCCGGGGGGGCTGTAGGA x64_19 GGTCGCA TGTCTG CAACGTACAACAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 39

1 NO: 38

NGSPIe TAGGTGC TGTGAC GCCGTGGGAGGGGTGGCCCGGCCCCCCCACGAGGACTA x64_20 GGTCGCA TGACTG CTCAAGAATTGCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 41

1 NO: 40

NGSPIe TAGGTGC TGTGAG GCCGACCCCGTGCGCTCGCTCCGCCGTCCCCCTCTGCAC x64_21 GGTCGCA ACTCTG GCGGACAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 43

1 NO: 42

NGSPIe TAGGTGC TGTGAG GTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTGGTCA x64_22 GGTCGCA TGTCTG TTTAGCGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 45

1 NO: 44

NGSPIe TAGGTGC TGTCTG CCAGGCATAGTTCACCATCTTTCGGGTCCTAACACGGAGC x64_23 GGTCGCA TCACTG CCATTACAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 47

1 NO: 46

NGSPIe TAGGTGC TGTGTG GGGTGCGTCGGGTCTGCGAGAGCGCCAGCTATCCTATAA x64_24 GGTCGCA AGTCTG GCGCCGTCCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 49

1 NO: 48

NGSPIe TAGGTGC TGTGTC GTTCGGTTCATCCCGCAGCGCCAGTTCTGCTTACCGTGC x64_25 GGTCGCA TCACTG CACAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 51

1 NO: 50

NGSPIe TAGGTGC TGTGTG GGATTCCGACTTCCATGGCCACCGTCCTGCTGTCTGAAAA x64_26 GGTCGCA AGACTG AATTTCTGCAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 53

1 NO: 52

NGSPIe TAGGTGC TGTCAC ACGCTCCAGCGCCATCCATTTTCAGGGCTAGTTGACGCTA x64_27 GGTCGCA AGACTG TGGCATCAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 55

1 NO: 54

NGSPIe TAGGTGC TGTGTC GCAGCGGCCCTCCTACTCGTCGCGGCGTAGCGTCCCATT x64_28 GGTCGCA TCTCTG GAGCAGTTGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 57

1 NO: 56

NGSPIe TAGGTGC TGTCAG ACCCTTCTCCACTTCGGCCTTCAAAGTTCTCGTTTCTAGA x64_29 GGTCGCA ACACTG GCCCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID SEQ ID NO: 59 1 NO: 58

NGSPIe TAGGTGC TGTCAC ACTCTCCCCGGGGCTCCCGCCGGCTTCTCCGGGATGTGA x64 30 GGTCGCA ACACTG TGGGAGTACCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 61

1 NO: 60

NGSPIe TAGGTGC TGTCAC GCCAGAGGCTGTTCACCTTGGAGACCTGCTGCGGACCGC x64 31 GGTCGCA TGTCTG TCACAAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 63

1 NO: 62

NGSPIe TAGGTGC TGTCTG CCCAGCCCTTAGAGCCAATCCTTATCCCGAAGTTATCAAT x64 32 GGTCGCA AGACTG CAGTTGCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 65

1 NO: 64

NGSPIe TAGGTGC TGTGTC GCTCCCCCGGGGAGGGGGGAGGACGGGGAGCGGGGTT x64 33 GGTCGCA AGTCTG GAGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 67

1 NO: 66

CCCCTGC

NGSPIe TAGGTGC TGTGAC CGCCCCGACCCTTCTCCCCCCGCCGCCGTATCTAAGGTC x64 34 GGTCGCA AGTCTG CC GTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 69

1 NO: 68

NGSPIe TAGGTGC TGTCTG GGCGGGGGGGACCGGCCCGCGGCCCCTCCGCCGCCGT x64 35 GGTCGCA AGTCTG CATGTCCAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 71

1 NO: 70

NGSPIe TAGGTGC TGTGAG GGATTCCCCTGGTCCGCACCAGTTCTAAGTCGGCTAGGG x64 36 GGTCGCA AGTCTG AAGGCAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 73

1 NO: 72

NGSPIe TAGGTGC TGTCAG GGCTACCTTAAGAGAGTCATAGTTACTCCCGCCGTGGCC x64 37 GGTCGCA TCTCTG CTTCACCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 75

1 NO: 74

NGSPIe TAGGTGC TGTGAG CACCTCTCATGTCTCTTCACCGTGCCAGACTAGAGGCGG x64 38 GGTCGCA TGACTG ATCCGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 77

1 NO: 76

NGSPIe TAGGTGC TGTCTC GCCCCTCGGGGCTCGCCCCCCCGCCTCACCGGGTCCGG x64 39 GGTCGCA ACACTG AACGCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 79

1 NO: 78

NGSPIe TAGGTGC TGTGTG GCCCTTCTGCTCCACGGGAGGTTTCTGTCCTCCCTAGTTT x64 40 GGTCGCA ACACTG GCCAGACCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 81

1 NO: 80

NGSPIe TAGGTGC TGTCTC GCTTGGCCGCCACAAGCCAGTTATCCCTGTGGTAATGATC x64 41 GGTCGCA TGTCTG TCTCTGAGTAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 83

1 NO: 82

NGSPIe TAGGTGC TGTGTG GCGGTTCCTCTCGTACTGAGCAGGATTACCATGGCGGCC x64 42 GGTCGCA TGTCTG AAATATAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 85

1 NO: 84

NGSPIe TAGGTGC TGTCTG CCGAGGCTCCGCGGCGCTGCCGTATCGTTCCGCCTATGG x64 43 GGTCGCA TCTCTG AGGAGGACAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 87

1 NO: 86

NGSPIe TAGGTGC TGTGAC AGGTCGTCTACGAATGGTTTAGCGCCAGGTTCCCCAGCC x64 44 GGTCGCA ACACTG TCAGGCAGTCTCGTACGGTTAAGAGCC SEQ ID NO: SEQ ID

SEQ ID NO: 89

1 NO: 88

NGSPIe TAGGTGC TGTCAC CATCTTTCCCTTGCGGTACTATATCTATTGCGCCAGCCTC x64_45 GGTCGCA TGACTG CCCTCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 91

1 NO: 90

NGSPIe TAGGTGC TGTCAG GACGGGTGTGCTCTTTTAGCTGTTCTTAGGTAGCTAGTAT x64_46 GGTCGCA AGTCTG CTCGTCCCCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 93

1 NO: 92

NGSPIe TAGGTGC TGTGTC G CTTTTAG GC CTACTATG G GTGTTAAATTTTTTACTCTCTC x64_47 GGTCGCA ACACTG TACAAGTCGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 95

1 NO: 94

NGSPIe TAGGTGC TGTCTG AGGGTGATAGATTGGTCCAATTGGGTGTGAGGAGTTCAA x64_48 GGTCGCA TGACTG CGTGTATTGTAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 97

1 NO: 96

NGSPIe TAGGTGC TGTGTC GACTTGTTGGTTGATTGTAGATATTGGGCTGTTAATTGTCA x64_49 GGTCGCA ACTCTG GTTTGTTGTAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 99

1 NO: 98

NGSPIe TAGGTGC TGTCTG GTAAGATTTGCCGAGTTCCTTTTACTTTTTTTAACCTTTCCT x64_50 GGTCGCA ACTCTG TATGCCAAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 1 01

1 NO: 1 00

NGSPIe TAGGTGC TGTCAG GCTGAACCCTCGTGGAGCCATTCATACAGGTCCCTGTCC x64_51 GGTCGCA AGACTG ACCGGCTAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 1 03

1 NO: 1 02

NGSPIe TAGGTGC TGTCAG GCTCGGAGGTTGGGTTCTGCTCCGAGGTCGCCCCACTTG x64_52 GGTCGCA TGACTG CATCCTTTGGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 1 05

1 NO: 1 04

NGSPIe TAGGTGC TGTGTC GGATTGCGCTGTTATCCCTAGGGTAACTTGTTCCGCAGAC x64_53 GGTCGCA AGACTG GTTGTGCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 1 07

1 NO: 1 06

NGSPIe TAGGTGC TGTCAG GCCTTATTTCTCTTGTCCTTTCGTACAGGGAGGAATTTGAA x64_54 GGTCGCA ACTCTG TATCTGTTTAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 1 09

1 NO: 1 08

NGSPIe TAGGTGC TGTCAG TTTCCCGTGGGGGTGTGGCTAGGCTAAGCGTTTTGCATTT x64_55 GGTCGCA TCACTG CATAGACCTTAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 1 1 1

1 NO: 1 1 0

NGSPIe TAGGTGC TGTCTC CAGGTGAGTTTTAGCTTTATTGGGGAGGGGGTGATCTACT x64_56 GGTCGCA ACTCTG GCATCGTTAGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 1 13

1 NO: 1 12

NGSPIe TAGGTGC TGTGTC GGCTCGTAGTGTTCTGGCGAGCAGTTTTGTTGATTTGAGT x64_57 GGTCGCA TGACTG CTAAGGAGTAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 1 15

1 NO: 1 14

NGSPIe TAGGTGC TGTGTG GTACTTGCGCTTACTTTGTAGCCTTCATCAGGGTTTGGGG x64_58 GGTCGCA TCACTG TTACCTGCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 1 1 7

1 NO: 1 1 6

NGSPIe TAGGTGC TGTGAG GTGACGGGCGGTGTGTACGCGCTTCAGGGCCCTGTACAA x64 59 GGTCGCA TCTCTG TCCTGGTAAGAAGTCTCGTACGGTTAAGAGCC SEQ ID NO: SEQ ID

SEQ ID NO: 1 19

1 NO: 1 18

NGSPIe TAGGTGC TGTCAC TCTTCATCGACGCACGAGCCGAGTGATCCACCGCTGTTTC x64_60 GGTCGCA AGTCTG CCTTCCAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 121

1 NO: 120

NGSPIe TAGGTGC TGTGAC GATCAATGTGTCCTGCAATTCACATTAATTCTCGCAGCTA x64_61 GGTCGCA AGACTG GCGTTCACAAAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 123

1 NO: 122

NGSPIe TAGGTGC TGTGTG GCTCAGACAGGCGTAGCCCCGGGAGGAACCCGGGGTCA x64_62 GGTCGCA TCTCTG CGAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 125

1 NO: 124

NGSPIe TAGGTGC TGTCTC GCCTACAGCACCCGGTATTCCCAGGCGGTCTCCCAAGGA x64_63 GGTCGCA TCACTG GCATATATAACAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 127

1 NO: 126

NGSPIe TAGGTGC TGTGAG GAGATCGGGCGCGTTCAGGGTGGTATGGCCGTAGAGCG x64_64 GGTCGCA ACACTG TTATCCAGTCTCGTACGGTTAAGAGCC

SEQ ID NO: SEQ ID

SEQ ID NO: 129

1 NO: 128

Name Region 1 Region 2

Capture

Probe64 ACATGCGACCGCACCTA TTTTTTTTTT\Biotin

plex CAGWSWSWS (SEQ ID NO: 130)

Table 2 - List of 256 precursor for the 256plex of in Fig. 8

Region

Name Region 3 4 Region 5

NGSPLEx TCGCGAAAT CTCAG TCGCCGCGTAAACGACGCGGCGCGCGTGCTGCCGCACT

256_1 TCGGTTGT ACAG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 132

NGSPLEx TCGCGAAAT GACAG AGCCCGCCGCGCACGCGCCCCTGCGCCCGCGCCGCCC

256_2 TCGGTTGT ACAG CAGGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 133

NGSPLEx TCGCGAAAT CTGTC CTGCTCCCGGCTGGGCCCACCGCCAAAGCAGCGGCCCC

256_3 TCGGTTGT TCAG ACAGGAGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 134

NGSPLEx TCGCGAAAT GTCAC CTCCTCCGGCCCCGCGCGCCCACTCCGCGCCCGGCCTG

256_4 TCGGTTGT ACAG GCCGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 135

NGSPLEx TCGCGAAAT CAGAC GCCGCCGCCGCCGCCGCCGCCGCCGCCCCGCTGCCTT

256_5 TCGGTTGT TCAG CTCAGCCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 136

NGSPLEx TCGCGAAAT CAGTG GCGGGTGGGCGGCCCGCGTTCCTTAGCCGCGGCTCCG

256_6 TCGGTTGT TGAG CGGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 137

NGSPLEx TCGCGAAAT GAGTC CCATTCCTGCCAGACCCCCGGCTATCCCGGTGGCCAGG

256_7 TCGGTTGT TCAG CTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 138

NGSPLEx TCGCGAAAT GACTC GGCCTCGCGTGCCTGGACAGCCCCGCGGGCCAGCAAG

256_8 TCGGTTGT TCAG CCTATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 139

NGSPLEx TCGCGAAAT CTCTC CCATCTCAGGGTGAGGGGCTTCGGCAGCCCCTCATGCT

256_9 TCGGTTGT TCAG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 140

NGSPLEx TCGCGAAAT CACAC GCGACCAAAGGCCGGCGCACGGCCTGGCCGCTCAGCG

256_10 TCGGTTGT AGTG ACTCCCGGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 141

NGSPLEx TCGCGAAAT GTGTG CGCGGCCTCAAAAGGCCTCCTAGGCCGCGGCGGGCAAA

256_1 1 TCGGTTGT TCAG GCACTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 142

NGSPLEx TCGCGAAAT GTGTC TACGCTCTCGCGCACCAGGTACGCCTGGTGTTTCTTTGT

256_12 TCGGTTGT AGAG GGTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 143

NGSPLEx TCGCGAAAT GAGAG CTCTGCCCAATCCCGGCTCCGGGCGACCCGGGCCCCTG

256_13 TCGGTTGT TGTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 144

NGSPLEx TCGCGAAAT CACTC TCTTGTAGGAGGCCCATTCCTCCCACCACGGGGCCACC

256_14 TCGGTTGT TGAG CACCCCGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 145

NGSPLEx TCGCGAAAT CTGAC CAGCCCCCAAACCCGACTGGTCGAAGGGGGACATCAAG

256_15 TCGGTTGT TGTG TCCCCCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 146

NGSPLEx TCGCGAAAT GTCTC CGCAGCCGACGCCGGCGCGAGAGCAGGGGCGGGGCCG

256_16 TCGGTTGT ACTG GCGCGGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 147

NGSPLEx TCGCGAAAT CAGTC GGCCCGCTCGGCAGGCCCCAACTGGCCCTCCCCCTTGG

256_17 TCGGTTGT AGAG CGGCGATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 148

NGSPLEx TCGCGAAAT GAGTG CGGGCCGAATGCCAGCCCGCCGAGCTCAGGGCAGCGG

256_18 TCGGTTGT TCAG GGAGCTGGTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 149

NGSPLEx TCGCGAAAT CACAG ACCTCCGCTGCGTCTCTCCGCGCCGCCGCCGCTGCTCG 256_19 TCGGTTGT TGAG CCGTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 150

NGSPLEx TCGCGAAAT CTCAC CGGCCCAGGTCTCGGTCAGGGCCAGGGCCGCCGAGAG

256_20 TCGGTTGT AGAG CAGCAGGATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 151

NGSPLEx TCGCGAAAT GTGAG CTGCCTCACGCATCACAGCACCCCCACCCGAGCGCGGG

256_21 TCGGTTGT AGTG CGGGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 152

NGSPLEx TCGCGAAAT CAGAC GGCCACGCTGCCACCAGCAGCAGGCCCATGGGGTGGCA

256_22 TCGGTTGT ACTG GGGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 153

NGSPLEx TCGCGAAAT CTGTG CCAGGGGTAGCCCCCTGGATTATGGTCTGACTCAGGACT

256_23 TCGGTTGT TCAG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 154

NGSPLEx TCGCGAAAT GTGTC CGAAGTTGCCCAGGGTGGCAGTGCAGCCCCGGGCTGAG

256_24 TCGGTTGT ACTG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 155

NGSPLEx TCGCGAAAT GACAC GTTACCTCCCCGCACACGGACTGTGTGGATGCGGCGGG

256_25 TCGGTTGT TCTG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 156

NGSPLEx TCGCGAAAT CAGAG AGAGCTCATGTATGGGTTAATCCGACCATGAGCTCTGTG

256_26 TCGGTTGT AGAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 157

NGSPLEx TCGCGAAAT CTGAG GGCCGGGCCACGGCCAGCATCCGGACCCGGGGCAGCG

256_27 TCGGTTGT ACAG GCGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 158

NGSPLEx TCGCGAAAT GACTC GCCATTACGGCTTCCCCGGCCAATAGACGCCCGGCTGC

256_28 TCGGTTGT ACAG CCTTACATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 159

NGSPLEx TCGCGAAAT CTCTG TTCAGCATTTCTGCTGAAATCTAGGGTGGAAATGCGTTCC

256_29 TCGGTTGT ACAG TAGTGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 160

NGSPLEx TCGCGAAAT CTCAC CTCAGCGGAAATCCGGCGATCTGGCCGGAAGTGCGGCA

256_30 TCGGTTGT TCAG CACTCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 161

NGSPLEx TCGCGAAAT GAGAC TGGCAGGAAGCTGCAGCCTTTCTCAAGAGCAGCCAGGAT

256_31 TCGGTTGT TCAG CTCCTGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 162

NGSPLEx TCGCGAAAT GAGTC GGTAGCGAGGAGAGCGGCTGAGGCTCAGTGCGCCTGC

256_32 TCGGTTGT TCTG GCGGCGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 163

NGSPLEx TCGCGAAAT GAGAG GCCATGGCAGCTTTCATGGCGTCTGGGGTTTTACCCCAC

256_33 TCGGTTGT TCAG TCATCTTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 164

NGSPLEx TCGCGAAAT CACTC TGCATCTTCAGGAGACGCTCGTAGCCCTCGCGCTTCTCC

256_34 TCGGTTGT ACAG TTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 165

NGSPLEx TCGCGAAAT CAGAG CCGTTGGCCACTTGTGGCCATTCCTACTCCCATGCCGGC

256_35 TCGGTTGT ACTG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 166

NGSPLEx TCGCGAAAT CTCAG AACTGGGTCCTACGGCTTGGACTTTCCAACCCTGACAGA

256_36 TCGGTTGT TGTG CCCGCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 167

NGSPLEx TCGCGAAAT GAGAC GCCGCTGCTGCAGCAGCTGCCTTATCCACCCGGAGCTT

256_37 TCGGTTGT TGAG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 168

NGSPLEx TCGCGAAAT GTCTC ACGCGGCCTCTCCCGGCCCCTTCCGTTTAGTAGGAGCC

256_38 TCGGTTGT TGAG GCACTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 169

NGSPLEx TCGCGAAAT CTCTG TGAGGGGCTTGGGCAGACCCTCATGCTGCACATGGCAG 256_39 TCGGTTGT AGAG GTGTATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 170

NGSPLEx TCGCGAAAT GTGTG CCCGGCCCAACAAACTACCTACGTCCGGGAGTCGCCAA

256_40 TCGGTTGT TGAG CCGACGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 171

NGSPLEx TCGCGAAAT GAGAC AGGCAGACTGCTGCAGGACGGGACTGGGCCGGGAACC

256_41 TCGGTTGT AGAG GGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 172

NGSPLEx TCGCGAAAT CACTG TCTAAACCGTTTATTTCTCCCCACCAGAAGGTTGGGGTG

256_42 TCGGTTGT TGAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 173

NGSPLEx TCGCGAAAT GAGTG AACACTGCCTTCTTGGCCTTTAAAGCCTTCGCTTTGGCTT

256_43 TCGGTTGT TGTG CATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 174

NGSPLEx TCGCGAAAT GTCAC CGGTAGCCGAAGGAGTTCAAAAGACCTCTAGTGCGCCCA

256_44 TCGGTTGT TCTG CCGCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 175

NGSPLEx TCGCGAAAT CTCTC GGTGACAGGGTGGCCCAGGAGCGGCCACTGAGATGAGA

256_45 TCGGTTGT TCTG CCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 176

NGSPLEx TCGCGAAAT GAGTG GATCACTCCCCAGGCGCTGAGGACGATGCCGCAGGCGG

256_46 TCGGTTGT TCTG CTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 177

NGSPLEx TCGCGAAAT GAGAG CTTAAGACCAGTCAGTGGTTGCTCCTACCCATTCAGTGG

256_47 TCGGTTGT TGAG CCTGAGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 178

NGSPLEx TCGCGAAAT GTCTG GAAGCTCCTAGAAGCTTCACAAGTTGGGGCACAACTCCT

256_48 TCGGTTGT TCTG GTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 179

NGSPLEx TCGCGAAAT CACTC TGGCGCGCGGCACTGGGAGCCGCCGGGCCGAGCCTGT

256_49 TCGGTTGT AGTG CAATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 180

NGSPLEx TCGCGAAAT GAGTC TCATAGAAAGAGAGGGAAGTTTTGGCGATCACAACAGCG

256_50 TCGGTTGT ACAG CCAAATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 181

NGSPLEx TCGCGAAAT CACTC CAAAGAATGCAAACATCATGTTTGAGCCCTGGGGATCAG

256_51 TCGGTTGT AGAG GGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 182

NGSPLEx TCGCGAAAT GTCTG GATAGCGCTCCTGTCTATTGGCTGCGCCATCGCCCGTCA

256_52 TCGGTTGT AGTG GACTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 183

NGSPLEx TCGCGAAAT CTCAG CATATGCAGGTCCCCTGTTGGCCATTCCAATGGGTGGCG

256_53 TCGGTTGT TGAG GTGGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 184

NGSPLEx TCGCGAAAT CTCAG ACTCCCTGCTCCTTGGGAATACGGACCACGCAGTCTATA

256_54 TCGGTTGT TCTG ATGCCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 185

NGSPLEx TCGCGAAAT CACAC CGGGGTAACCGTGGAGGGCGACGCGCAGAGGCTGCGG

256_55 TCGGTTGT TGTG CTATTTATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 186

NGSPLEx TCGCGAAAT CAGTC GGACTGGTCCCATGAGGCAGAAGGAGCACCAGCGCCTG

256_56 TCGGTTGT TGTG CTGGGTGGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 187

NGSPLEx TCGCGAAAT GTCTC CGAGAAACAGCGCCCGACACCTGGCCCTTCGCAGCTCT

256_57 TCGGTTGT TGTG CGCCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 188

NGSPLEx TCGCGAAAT CAGAG CTGCATGGCCTTCATGACATGAAGGTTGGGCACATTCTT

256_58 TCGGTTGT TGTG GTCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 189

NGSPLEx TCGCGAAAT CTGAC CCGAAACCCATGGTGTCGGCTGTATCCGAGAGCTGGGG 256_59 TCGGTTGT TGAG AGCAGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 190

NGSPLEx TCGCGAAAT GACAC TTCATGCAGATCACCTGCACCCCGCTTGTGTTCAGTGGG

256_60 TCGGTTGT TCAG GTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 191

NGSPLEx TCGCGAAAT GAGAC AGCTGCCGGGGTCCGGTTCCTCAGCTCCAGGTGGATCC

256_61 TCGGTTGT ACAG TTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 192

NGSPLEx TCGCGAAAT CTCTG CTCGGAAGTAGCCCCCGTAGGTGCCCTGCTTGTGGTCAA

256_62 TCGGTTGT TGAG ACTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 193

NGSPLEx TCGCGAAAT CACAC CGGGGGTAGCGGTCAATTCCAGCCACCAGAGCATGGCT

256_63 TCGGTTGT AGAG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 194

NGSPLEx TCGCGAAAT GTCAC GAAACATGATCGCTTATAAGCCAGCGGTCCCAATTCGGT

256_64 TCGGTTGT ACTG CCACCGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 195

NGSPLEx TCGCGAAAT GTGTG CCCACACGTCCATGACTGGTCGTCCTAGATTTTAGGTGT

256_65 TCGGTTGT ACAG CTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 196

NGSPLEx TCGCGAAAT CACAC CTTTAGCTCGAGATTGTCCCTCTCTGTCCAGCAGATAGG

256_66 TCGGTTGT ACAG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 197

NGSPLEx TCGCGAAAT CTCTC GCCGTCCTGCGCAAGCGCTTTTCAACCCCACTCCTTTCT

256_67 TCGGTTGT AGAG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 198

NGSPLEx TCGCGAAAT GACTG CAGTCTCTGGGAGAATGGGCAGTTCCCAATCTTGGCCCC

256_68 TCGGTTGT TGTG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 199

NGSPLEx TCGCGAAAT GTCTG GGTTGGTGCTTGCCACACTTCTTACAGAAAGTCCGGCGG

256_69 TCGGTTGT TGTG GTTTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 200

NGSPLEx TCGCGAAAT GTGTC CATGTAGTTGAGGTCAATGAAGGGGTCATTGATGGCAAC

256_70 TCGGTTGT ACAG AATATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 201

NGSPLEx TCGCGAAAT CTCAC CGGAACTGGAGGTTTCCTTTTCCGCCATAGTTTGTCCTG

256_71 TCGGTTGT TGTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 202

NGSPLEx TCGCGAAAT GTGTC TTGTAGTCTGAGAGAGTGCGGCCATCCTCCAGCTGTTTG

256_72 TCGGTTGT TCAG CCGGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 203

NGSPLEx TCGCGAAAT GTCAG CTGCAGTGGCTTTAAACCCACAGTAGTAACCTGCAGGAT

256_73 TCGGTTGT TGTG CACACTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 204

NGSPLEx TCGCGAAAT CACTG CGAAGGACAGGTGGTCTCTTCGTTGGGACGTCCCCTTTG

256_74 TCGGTTGT TCTG CCAGCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 205

NGSPLEx TCGCGAAAT CAGTG AGCTTGGCTCCCTTCTTGCGGCCCAGGGGCAGCGCATG

256_75 TCGGTTGT AGAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 206

NGSPLEx TCGCGAAAT GTCTG TTCCGACATGTCCGCATTTTTGATCACGGCCTTTCGGTG

256_76 TCGGTTGT TGAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 207

NGSPLEx TCGCGAAAT CAGAG CTTGGGAAGACCAAGTCCTCAAGGATGGCATCGTGCACA

256_77 TCGGTTGT TCAG GCTGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 208

NGSPLEx TCGCGAAAT CTCTG CGGCCGCCTCCAGGAACGCCGACCACTCCACTTTAGGT

256_78 TCGGTTGT TCAG ATCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 209

NGSPLEx TCGCGAAAT GTCTC AGGCGAAGTTCCGTCTACGGCTATTTAATGGAGCGCCTG 256_79 TCGGTTGT TCTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 210

NGSPLEx TCGCGAAAT CTCTC TGTATGTTCCATCCATGTGAGCAGCAAATGTGTATTTCCC

256_80 TCGGTTGT TGAG ACTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 21 1

NGSPLEx TCGCGAAAT CAGTC GGTCTCATCCGAACCCTGCGGATATATTTTTCACCCAAGA

256_81 TCGGTTGT TGAG AATTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 212

NGSPLEx TCGCGAAAT GACAC GGCCTTCACGCGGCCCAGGAGTTTCTTATTGTTGCGGCA

256_82 TCGGTTGT AGAG GTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 213

NGSPLEx TCGCGAAAT GTGAG CCTTTTCCAAGGATTTTACGTTGCGGCTTGTTAGGGTGAT

256_83 TCGGTTGT ACAG TTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 214

NGSPLEx TCGCGAAAT GTGTG ACTGTTCTCTCTTGGCAAAGTAATCAGGATACATTGCCTG

256_84 TCGGTTGT AGAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 215

NGSPLEx TCGCGAAAT GTCAC ACAGTAGCATGCAGTCCCACAACTTGTACCAGCATCCCC

256_85 TCGGTTGT TGTG AGCGTCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 216

NGSPLEx TCGCGAAAT GTCAG ACTTGGCTCCAGCATGTTGTCACCATTCCAACCAGAAATT

256_86 TCGGTTGT TCAG GGCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 217

NGSPLEx TCGCGAAAT GACTC ACAATGCAAAGATGGCTTTTCAGAGCAGCCAGTGGGGGT

256_87 TCGGTTGT AGAG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 218

NGSPLEx TCGCGAAAT CTGAG CGACGTAGCCCGGCCTCTTCGACCTGCACCTCCGCGGC

256_88 TCGGTTGT ACTG TCCCTCTGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 219

NGSPLEx TCGCGAAAT CTCTC AGTGGAAACAGGATTACTATGATACAAAACTTCCACTACT

256_89 TCGGTTGT ACTG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 220

NGSPLEx TCGCGAAAT GTGAC GCAAAGGCAATCTTCAAATAGAAGCTGGCAACACAAGAC

256_90 TCGGTTGT ACAG CTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 221

NGSPLEx TCGCGAAAT GTGTG AATCACGCACTGTCCCCAACAGCCCCAGTTAACACAGGG

256_91 TCGGTTGT TGTG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 222

NGSPLEx TCGCGAAAT CTCAG CAGGGTTTCTGGTCCAAATAGGCTTGGTCTTGTTTATGGT

256_92 TCGGTTGT ACTG CGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 223

NGSPLEx TCGCGAAAT GTGTC CCCGAATCCGCCGGCCCTTCTCACCAAGAACATTCTGTT

256_93 TCGGTTGT TCTG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 224

NGSPLEx TCGCGAAAT GTCTC GGAGATCCATCATCTCTCCCTTCAATTTGTCTTCGATGAC

256_94 TCGGTTGT TCAG ATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 225

NGSPLEx TCGCGAAAT GACTC TGGCATTAGCAGTAGGTTCTTGTATTTGAGTCTGCTTGGT

256_95 TCGGTTGT ACTG CGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 226

NGSPLEx TCGCGAAAT CTCAC GAAAACTGGTCAGATGAATATTATTGCTTCCCATTTTCAA

256_96 TCGGTTGT AGTG CCAGTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 227

NGSPLEx TCGCGAAAT GACTC TCCAGAGGGTCCGGATCGCTCTCTTCTGCACTGAGGTTG

256_97 TCGGTTGT TGTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 228

NGSPLEx TCGCGAAAT CTCTG CCATCCTGGCAGGCGGCTGTGGTGGTTTGAAGAGTTTG

256_98 TCGGTTGT ACTG GACTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 229

NGSPLEx TCGCGAAAT GTCAG GCTGGCAAGGCTGAGCAATTCATGTTTATCTGCAACAGC 256_99 TCGGTTGT TGAG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 230

NGSPLEx TCGCGAAAT GAGAG AGCATCAGCTACTGCCAGCGGTTCATGGGCTTCTTTTAC

256J 00 TCGGTTGT ACTG TATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 231

NGSPLEx TCGCGAAAT CAGTC TGAGTGAGCCCTCCTGCCACGTCTCCACGGTCACCACCT

256J 01 TCGGTTGT TCTG CCTCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 232

NGSPLEx TCGCGAAAT GTGAC CTTTGGGTCCCAAGGTGCTCTTTACCAAGTCTCCAATGG

256_102 TCGGTTGT AGAG CGATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 233

NGSPLEx TCGCGAAAT CAGTG AACCCAATTAGTTCCCAGAAGTCACAACTCAGCTCATGG

256_103 TCGGTTGT ACTG CCACCTGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 234

NGSPLEx TCGCGAAAT CACAC CTCCTTGGTTCCATCTCCCGTGGCATCGCTTCCCTCTCG

256_104 TCGGTTGT TGAG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 235

NGSPLEx TCGCGAAAT GAGTG TATTTCACCAGGCCGGCAAAGAATGGACGGTCCTTCAGG

256_105 TCGGTTGT AGAG TCAACGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 236

NGSPLEx TCGCGAAAT CTGTG CCCTCAGAGGACAGGGCGCGGTTGCTGGGTCATGAGCA

256J 06 TCGGTTGT TCTG CCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 237

NGSPLEx TCGCGAAAT CTGTC CAGGCACCAGACCAAAGACCTCCTGCCCCACAGCAAATG

256_107 TCGGTTGT TGTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 238

NGSPLEx TCGCGAAAT CAGTG AGTTTCCTCTTCACTCAGCAGCATGTTGGGGATCCCGCG

256_108 TCGGTTGT AGTG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 239

NGSPLEx TCGCGAAAT GTCAC TCTGTGAGAAAACCTTGGAGAATCAATAATGGTGGATTCA

256J 09 TCGGTTGT TGAG TTGATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 240

NGSPLEx TCGCGAAAT GAGAC GCTGTATCTGGTCCTGGCGGCCGGCTGTGATGTTTGACA

256_1 10 TCGGTTGT ACTG TTGTCCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 241

NGSPLEx TCGCGAAAT CACTC GATGGCGGCGGGGGGCAGGGGGCGCACGTAGCCTGGC

256_1 1 1 TCGGTTGT TCTG CATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 242

NGSPLEx TCGCGAAAT GACTG CTCTCTCTTCAGCAATGGTGAGGCGGATACCCTTTCCTC

256_1 12 TCGGTTGT TGAG GGGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 243

NGSPLEx TCGCGAAAT GTGTG TCTGGGACAAGACAGTCGAGGGAGCTTCTTCCTCAGGGA

256J 13 TCGGTTGT ACTG ACTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 244

NGSPLEx TCGCGAAAT CAGAG GCTGCCAAAGCTGGGTCCATGACAACTTCTGGTGGGGC

256_1 14 TCGGTTGT AGTG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 245

NGSPLEx TCGCGAAAT GTCAG AGACTGCCAGCGAAGCCCCTCTTATGAGCAAAAGAGCAA

256J 15 TCGGTTGT TCTG CCCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 246

NGSPLEx TCGCGAAAT CACTC CTTCAGGTGTCCTTGAAGCAATAATTTCTGTCAGTACTTT

256_1 16 TCGGTTGT TCAG TTCATTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 247

NGSPLEx TCGCGAAAT CTGTC AACGATCAAAATTAGACATGTCTTCATCTGAATCATCTTC

256_1 17 TCGGTTGT ACAG CCAGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 248

NGSPLEx TCGCGAAAT GTGAG CGGCCACACCATCTTTGTCAGCAGTCACATTGCCCAAGT

256J 18 TCGGTTGT TGTG CTCCAACTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 249

NGSPLEx TCGCGAAAT GAGTC TGACCTCTCACTTTTCCAGCACGGGCCAGGGAACCATGG 256J 19 TCGGTTGT AGTG ACTTTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 250

NGSPLEx TCGCGAAAT GTGAC GTCAGCATAATCTTTATTTCAAAATAACATTTTTATTATGG

256J 20 TCGGTTGT TGAG TCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 252

NGSPLEx TCGCGAAAT CTCAC AGCCACAGGATGTTCTCGTCACACTTTTCCATGTAGGCG

256_121 TCGGTTGT ACAG TTATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 253

NGSPLEx TCGCGAAAT GTCTG TATCCGTTCCTTACATTGAACCATTTTACTGTTCCCAAAAC

256J 22 TCGGTTGT ACAG CTTCGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 254

NGSPLEx TCGCGAAAT CTGTC GACTTTTACAATCGATTCCCCAAACCCCTTTATGGCAGCA

256_123 TCGGTTGT AGTG ACACTGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 255

NGSPLEx TCGCGAAAT GACAC TTCTTGTAATTTGCATAATCCTCAAGAATGGAATCCACTG

256_124 TCGGTTGT TGAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 256

NGSPLEx TCGCGAAAT GAGAG TGGTCCCAGGGGAAAGGAAGAGGCCAGTTGGTCCAGTT

256J 25 TCGGTTGT AGAG TTGATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 257

NGSPLEx TCGCGAAAT GACTG CAGGGGACGGTACTCCACATCCTCTCTGAGCAGGCGGT

256_126 TCGGTTGT ACTG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 258

NGSPLEx TCGCGAAAT CTCTG CTTCTACATCATCAGCTGCCATACGAAGAAGGGACTCCG

256J 27 TCGGTTGT AGTG TTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 259

NGSPLEx TCGCGAAAT GACTG GCCACCAGCATCAACCTTCTTGGCTTCGGGTTTCTTCTG

256_128 TCGGTTGT ACAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 260

NGSPLEx TCGCGAAAT GTCTG CCCCACCGGTGCTCTTGGTACGAAGATCCATGCTAAATT

256_129 TCGGTTGT AGAG CCCCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 261

NGSPLEx TCGCGAAAT CAGAG TTGTATATAAGATTACTTTATTCCTGCATCTTCTCAATGGT

256J 30 TCGGTTGT TGAG TTCTTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 262

NGSPLEx TCGCGAAAT GACAG CATTTTCATGGTTTTGTAGAGCTTCAATTTTGTCTAAGCCT

256_131 TCGGTTGT ACTG CCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 263

NGSPLEx TCGCGAAAT GTGAC CATAGTTGTCAACAAGCACAGTGAAAGCGCCATTCTCTTT

256J 32 TCGGTTGT TGTG ACATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 264

NGSPLEx TCGCGAAAT GTCTC GCCAACAGCATGCTGGGTAACATTGTAGACTCTTCCTGG

256_133 TCGGTTGT AGTG TCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 265

NGSPLEx TCGCGAAAT CTGAC AGGAGATCTCCACAGGGGCTGGACGGTTCATTATGGCAA

256_134 TCGGTTGT AGAG ATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 266

NGSPLEx TCGCGAAAT CTGAG TTGAGCATCTCGTAGTTGGGAGGCTGGCCGCTGTTGACA

256J 35 TCGGTTGT TCAG GGAGAGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 267

NGSPLEx TCGCGAAAT CAGAC CTCCCTTTCCCCAGTAGTTTCGGTTTCTCAACAGTTTCCT

256_136 TCGGTTGT TGTG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 268

NGSPLEx TCGCGAAAT GTGAG TGGTTTTTAACAGGTTTAACCAATCATCTACTATCTGATTG

256J 37 TCGGTTGT ACTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 269

NGSPLEx TCGCGAAAT CTGAC CCTTCTGTCCTCATGTTGGCAGAGATATCTACTCTGTGGT

256_138 TCGGTTGT AGTG CGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 270

NGSPLEx TCGCGAAAT GACAG GGATTCCAGTAGCCAGGTTGGTACGGGACGGCATCATAA 256J 39 TCGGTTGT AGAG CATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 271

NGSPLEx TCGCGAAAT CTGAG CTTCAGCGGAGGCATTTCCACCAATGAGCGAGTCATTGG

256J 40 TCGGTTGT AGTG TCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 272

NGSPLEx TCGCGAAAT GTCAG GTTTATGGTAAAGCTTAGCCTTCAGACCAATCATTTTCTTT

256_141 TCGGTTGT ACAG GCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 273

NGSPLEx TCGCGAAAT CTCAC GGCCGCAAAAGGGAAGAGAACTACACGCTGCTTCCGGT

256J 42 TCGGTTGT TCTG TCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 274

NGSPLEx TCGCGAAAT GAGTG TTGTTCACTGGGTCTTTGTCTTTCTTGGCCGACTTTCCAG

256_143 TCGGTTGT TGAG CGTCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 275

NGSPLEx TCGCGAAAT GAGAC CTTCCCAGTTAAGGCTCTTTATTTTATTTTGAACACTTTTT

256_144 TCGGTTGT AGTG TGCATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 276

NGSPLEx TCGCGAAAT CAGAC ATCAACAAGCCACGGTTTTAGCTCTTCAGGAATCTTTACT

256J 45 TCGGTTGT ACAG TTAACTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 277

NGSPLEx TCGCGAAAT CACAC GGTGGTTCCTTGAGGGCTTTGATGATCAGGGCAGAGGC

256_146 TCGGTTGT TCTG AGAAGGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 278

NGSPLEx TCGCGAAAT GTGTC AATTCACACACCTCACAGTAAACATCAGACTTTGCTGGGA

256J 47 TCGGTTGT AGTG CCTCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 279

NGSPLEx TCGCGAAAT GTCTG TGTCATCCTTCTTGCCACCTCCAGGACCATGACCACCAC

256_148 TCGGTTGT ACTG TCTGACTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 280

NGSPLEx TCGCGAAAT GTGAG GAGCAAGGAGGGCTGGAAGCTGTTAGTCAGAGTGTTGA

256_149 TCGGTTGT TCTG AGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 281

NGSPLEx TCGCGAAAT CACAC AAAATTGTGCGGATGTGGCTTCTGGAAGACCTTCATTCTA

256J 50 TCGGTTGT TCAG AAGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 282

NGSPLEx TCGCGAAAT CACTG GCAGTTTTCTAATTGAGAATGTAATCTTGGTCTTTAAAGA

256_151 TCGGTTGT TGTG ACATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 283

NGSPLEx TCGCGAAAT CACAC GTTTCTGCATCAGCCCGCTCATCAAATCCAGGGAAGTTG

256J 52 TCGGTTGT ACTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 284

NGSPLEx TCGCGAAAT CTGAG CCAGCGGCAACCTCAGCCAAGTAACGGTAGTAATCTCCT

256_153 TCGGTTGT TCTG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 285

NGSPLEx TCGCGAAAT GACAC GTGATCGGGGTTTCTTGATACCATTTCTGTGCCATTTTCG

256_154 TCGGTTGT TGTG GGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 286

NGSPLEx TCGCGAAAT GTCAC AGGAGGTCCTGCTGAGTTGGTGAATCTCTGGTAACGGTG

256J 55 TCGGTTGT AGAG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 287

NGSPLEx TCGCGAAAT CTGAG CCTGGTTTTCTAAAATTCTTCAGGTCAATAGTCAAGCCTT

256_156 TCGGTTGT TGAG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 288

NGSPLEx TCGCGAAAT CTGAC ACAATATCACCTTTCTTATAGATTCGCATATATGTGGCCA

256J 57 TCGGTTGT ACAG AAGGATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 289

NGSPLEx TCGCGAAAT CTCTG AAACAAAACAAGAAAAAGTAATCTGCTAAAAACTATAGGG

256_158 TCGGTTGT TGTG TCCCCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 290

NGSPLEx TCGCGAAAT CAGTG TAGAATCTTTTTTATTCAGAAAAAAAAAACCCCAAAAAACA 256J 59 TCGGTTGT ACAG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 291

NGSPLEx TCGCGAAAT CTGTG AGTTTAATAAATACAAATACTCGTTTCTTTTTGATTAGTGT

256J 60 TCGGTTGT AGAG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 292

NGSPLEx TCGCGAAAT GTGAC ACTTTGAGATTCTTTTCTTTTGCGCCTCTTATCAAGTCAG

256J 61 TCGGTTGT TCAG CTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 293

NGSPLEx TCGCGAAAT CACAG AGCCTGGTTGGAGGATTCCTAGTTTTATACATGAGAAATA

256_162 TCGGTTGT AGAG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 294

NGSPLEx TCGCGAAAT GAGTC GTTCCCAAGATAGAAGAGTAGGTATGAAGCAATTCTGAC

256_163 TCGGTTGT AGAG TCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 295

NGSPLEx TCGCGAAAT GACTC TCTTTGTATGAGTCTTCATTCAGTGTATCAAGTTCATGGT

256_164 TCGGTTGT AGTG CGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 296

NGSPLEx TCGCGAAAT CTGTG ACAGATGAATGTAGGATTGATGCAAGTCACTTCCAGGAA

256_165 TCGGTTGT ACTG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 297

NGSPLEx TCGCGAAAT GTGAG TCGCTTTTAGCTCCTCGAGTTTCTTCTGCTCCTCTTTTTGT

256J 66 TCGGTTGT TGAG TTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 298

NGSPLEx TCGCGAAAT CTCAG TACTTCTGGGCCGTCACAGGGGAGGGCAGGTGGATGGT

256_167 TCGGTTGT AGTG GATCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 299

NGSPLEx TCGCGAAAT CACTC TGGTGCCGGATGAACTTCTTGGTTCTCTTTTTGACGATCT

256_168 TCGGTTGT ACTG TGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 300

NGSPLEx TCGCGAAAT GAGTC TATGCCTAGAACTTTCACGCCAATTATTTCACCTCTTGCA

256J 69 TCGGTTGT TGAG CATATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 301

NGSPLEx TCGCGAAAT CACAG CCCAGGTCCTGTGATGTTTATTGAAGGAAGCAAGGGCAG

256_170 TCGGTTGT TCTG GGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 302

NGSPLEx TCGCGAAAT CAGAC GTCCCTTTGTTTTCTTCTTCTTTTTCCCCACTCTAGTTGGT

256J 71 TCGGTTGT AGTG ACAGGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 303

NGSPLEx TCGCGAAAT CAGAC CCATCGATGTTGGTGTTGAGTACTCGCAAAATATGCTGG

256_172 TCGGTTGT TCTG TCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 304

NGSPLEx TCGCGAAAT GTGTG GATACCACAGAATCAGCAGGGTGAGAAACAATTGCACAA

256_173 TCGGTTGT TCTG AAGACTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 305

NGSPLEx TCGCGAAAT GACTC AAAGCTTGAGATCACTTGAGGCCAGAGTTTTCAGACCTG

256_174 TCGGTTGT TCTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 306

NGSPLEx TCGCGAAAT GACAG CGGGTGTGGACGGGCGGCGGATCGGCAAAGGCGAGGC

256J 75 TCGGTTGT TCTG TCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 307

NGSPLEx TCGCGAAAT CACAG CAGCGCGAGTGCAGAGCATGGTGGTAGATGTGGCAGAG

256_176 TCGGTTGT ACAG GATGGCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 308

NGSPLEx TCGCGAAAT CTGAG GACCCTCATAGACAGCAGACAGAAGAGGAGTAATATGAT

256_177 TCGGTTGT TGTG GTTTATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 309

NGSPLEx TCGCGAAAT GTGTG AAATACACTTTTAATTGATTTCAGATAAAAACTACTTGGTC

256J 78 TCGGTTGT AGTG GGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 310

NGSPLEx TCGCGAAAT GTGAG GCCGCGCGAAGCCGGAGAGGAGAAGAAGAGAAGGAGG 256J 79 TCGGTTGT TCAG GTTAGGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 31 1

NGSPLEx TCGCGAAAT CAGTC CAAATCTCAGGGAAGCAGTGATGGAGGACACAATCTGGC

256J 80 TCGGTTGT ACTG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 312

NGSPLEx TCGCGAAAT CACTC GGTCATAGTGGAGGGTAAGAGCTTTTACATCCCGCAGTG

256_181 TCGGTTGT TGTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 313

NGSPLEx TCGCGAAAT CACAG GATCCATCATTTCTCCTTTAAGCTTATCTTCCAAAATGGTC

256J 82 TCGGTTGT TGTG GGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 314

NGSPLEx TCGCGAAAT GTCTG TGTTTACTGATTTCTGTCTGGTTAAACATCCAATACTGGT

256_183 TCGGTTGT TCAG CGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 315

NGSPLEx TCGCGAAAT GACAG GCCTATCTCTTTCCATCAGACTCCAGTGATACCCAATGGT

256_184 TCGGTTGT TCAG CGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 316

NGSPLEx TCGCGAAAT GACAG CCTGTAATCTCAGCACGTTGGGAGGCGAGGTGGGTGGA

256J 85 TCGGTTGT AGTG TGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 317

NGSPLEx TCGCGAAAT CTCTC CTTAAATACCAGATACATTTTTAGTCCTCTACATAATGGTC

256_186 TCGGTTGT ACAG GGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 318

NGSPLEx TCGCGAAAT GACAC GTTTTTTGGAAGATTCGGGTTCAGCACAGGATTCCATTTG

256J 87 TCGGTTGT AGTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 319

NGSPLEx TCGCGAAAT GTCTC TGATATCCTTGTTTTTAACTGTTGTGGCTTGCTGAATCAA

256_188 TCGGTTGT AGAG ATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 320

NGSPLEx TCGCGAAAT CAGAG AAGACGGAGTAGTTAAGAGCCAGGCCTAATCGGATGGTG

256_189 TCGGTTGT ACAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 321

NGSPLEx TCGCGAAAT CTGTC CGAGTTCCAGAGACAATATCAAAATTACCCTCCTTTTGGT

256J 90 TCGGTTGT ACTG CGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 322

NGSPLEx TCGCGAAAT CTCTG GGTGAGAGAACTAATAGCAACCAGGCAACTGAGGACGAA

256_191 TCGGTTGT TCTG GTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 323

NGSPLEx TCGCGAAAT CTCAC TTGGGGTGCTTTATCTTCTTTGAGTTTTCGCACAAGATGG

256J 92 TCGGTTGT ACTG TCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 324

NGSPLEx TCGCGAAAT CTGTG GGGCATGGGCTCACATTCACTTCCTTTATAACTCCATCCT

256_193 TCGGTTGT TGTG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 325

NGSPLEx TCGCGAAAT CAGTC GCCTGTTTTCCCTTTGCTCCCCTTTTCCCTTTTGTTTGCA

256_194 TCGGTTGT TCAG CTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 326

NGSPLEx TCGCGAAAT GAGAC CATTTTTCCGATAGTTAATAGTAATGGAGTAATAATGTTG

256J 95 TCGGTTGT TCTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 327

NGSPLEx TCGCGAAAT GTGAC CACTTGGCCCTTTCTCTTCTTATCTCCTCCCAGTTCTGGT

256_196 TCGGTTGT TCTG CGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 328

NGSPLEx TCGCGAAAT GACTG TCGTCCTCCTCCTCTTCATCCACACCATCCACCTCGGTG

256J 97 TCGGTTGT TCAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 329

NGSPLEx TCGCGAAAT GACAG CCTCCTCTTCCTCCCCACCTTCTTCCTCTTCTTCGTCTAC

256_198 TCGGTTGT TGAG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 330

NGSPLEx TCGCGAAAT GACTG ACGATGGCGGAGAAAGGAAGAGGAGGGAAGCTGGCGG 256J 99 TCGGTTGT AGAG AATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 331

NGSPLEx TCGCGAAAT GAGAG TATAATACAAAAAAAGACCAAAAAACAAAACAAAACAAAA

256_200 TCGGTTGT ACAG CATCAATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 332

NGSPLEx TCGCGAAAT CTCAC AACACAAGTGTGTTGTTGTCTTCTATCTTCTTCATGGCAT

256_201 TCGGTTGT TGAG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 333

NGSPLEx TCGCGAAAT GAGTG ACCAAAACCACAATTTCTGCAGTTTAAAATGTTTCACTTG

256_202 TCGGTTGT ACAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 334

NGSPLEx TCGCGAAAT CACAG CCGCTGCTCGGTCCCCCAGGCCCCGCCGTCCTTGCTGT

256_203 TCGGTTGT AGTG TTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 335

NGSPLEx TCGCGAAAT GACTG CGAGATCCTGGTGCTCCCACTCGCGTTGCTGCAGCAAGA

256_204 TCGGTTGT AGTG AATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 336

NGSPLEx TCGCGAAAT CACTG GCCGGCCGGGGTGGGGAACGAGCGCCGGGTTCCGTCC

256_205 TCGGTTGT TCAG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 337

NGSPLEx TCGCGAAAT GTCAG TCTCTGCCACCGCTGGTGCTGCTGTCTCCCACTCGGTGG

256_206 TCGGTTGT AGTG TCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 338

NGSPLEx TCGCGAAAT CTGTG CATCGAAGACGCTCGCTTCAGAAATGTCCCTGACTGCTG

256_207 TCGGTTGT AGTG CGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 339

NGSPLEx TCGCGAAAT CTGAC GTCTTTCAGGTCAATGTAGTGCTGCTTCAGGTGTTCTTCA

256_208 TCGGTTGT ACTG GAGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 340

NGSPLEx TCGCGAAAT CTGTC CATCAGCATAGCCTCCGATGACCATGGTGTTCCACAAAG

256_209 TCGGTTGT TGAG GGTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 341

NGSPLEx TCGCGAAAT CACTG GATGCCCAGAATCAGGGCCCAGATGTTCAGGCACTTGG

256_210 TCGGTTGT ACTG CGGTGGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 342

NGSPLEx TCGCGAAAT CAGTG AGAACCGGAAGAGAAAGGGGCTGCGGTGCAGCACGGGA

256_21 1 TCGGTTGT TCAG AATAGGGTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 343

NGSPLEx TCGCGAAAT GTGAG CCCCCCAACCCTCACTGTTTCCCGTTGCCATTGATGGTG

256_212 TCGGTTGT AGAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 344

NGSPLEx TCGCGAAAT CACTG ACCTCATAGGTGCCTGCGTGGGCGCTCTTGTGGTCCAG

256_213 TCGGTTGT ACAG GCTCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 345

NGSPLEx TCGCGAAAT GAGAC ACAGGAGTCTTGCCCAAGCCCTGTCATGTCAGTGTGTGT

256_214 TCGGTTGT TGTG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 346

NGSPLEx TCGCGAAAT CAGTG CTTCTTCAAGGTGATATAGACGCTGCCCGACGTCCGGTG

256_215 TCGGTTGT TCTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 347

NGSPLEx TCGCGAAAT GTGTC GCCATCTGGGCCATCAGACCTGGCTGCCGGGGCGCATG

256_216 TCGGTTGT TGAG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 348

NGSPLEx TCGCGAAAT GACTC GCTTCTTGGGAAATGAAGCCACAGCCAGCTCATATATGT

256_217 TCGGTTGT TGAG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 349

NGSPLEx TCGCGAAAT CAGAG CTCATCCACGATGGCTGCTATCGGTAAACAGTTAAAACA

256_218 TCGGTTGT TCTG GTCTGTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 350

NGSPLEx TCGCGAAAT GTCAG TGACCCGCTCGATCGGAGCCACGGCCGTCTTGGAGATG 256_219 TCGGTTGT AGAG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 351

NGSPLEx TCGCGAAAT CAGTC GGGTGATCAGCTGTGAGGCATTGAACTTGGCCACCACAC

256_220 TCGGTTGT AGTG TCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 352

NGSPLEx TCGCGAAAT GTGTC ACAGCACAGTAACAAAGTTATTAGGAAAACAGGACTACC

256_221 TCGGTTGT TGTG ACAAAGATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 353

NGSPLEx TCGCGAAAT CAGAC ACCAATGTTTTTTAGAATAGTGGCACCATCATTGGTTGGT

256_222 TCGGTTGT TGAG CGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 354

NGSPLEx TCGCGAAAT GACTG GCAGTTTACGCTGTCTAGCCAGAGTTTCACCGTAAATATG

256_223 TCGGTTGT TCTG ATTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 355

NGSPLEx TCGCGAAAT GTCTC CACTCTTTCACTTAAAGAGATATAGCTAGAAGGATTCACA

256_224 TCGGTTGT ACAG GTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 356

NGSPLEx TCGCGAAAT GACAC ACCTTCAGGTCGTCCAGCTGTTTCAGCAGCTCCTCCTGG

256_225 TCGGTTGT ACAG TCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 357

NGSPLEx TCGCGAAAT CACAG GCCGTCAACTTGCGTCGGAACATGGTCCCCGCTTCTCGC

256_226 TCGGTTGT TCAG TCTGGTCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 358

NGSPLEx TCGCGAAAT GTGAC CGATCCAAAAAGTGCGCGATGCGAGTAGTCAAGTCGTAC

256_227 TCGGTTGT ACTG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 359

NGSPLEx TCGCGAAAT CTGTC AGACAATGGTCCCTCTATTTCAACACCTTTTTCGGTGACA

256_228 TCGGTTGT TCTG GTGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 360

NGSPLEx TCGCGAAAT GAGTC GGTGATCTTGCTCTTGCTCCTTTCGATGGTCACCACCCC

256_229 TCGGTTGT ACTG TCCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 361

NGSPLEx TCGCGAAAT CTCTC AACAGCCTTTAGTTCTACAGGAAATGGCACTGATGGACA

256_230 TCGGTTGT TGTG GAAGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 362

NGSPLEx TCGCGAAAT GAGAG CCGGCTGTCTGTCTTGGTGCTCTCCACCTTCCGCACCAC

256_231 TCGGTTGT TCTG CTCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 363

NGSPLEx TCGCGAAAT CTGAG GGGAGGTGAACCCAGAACCAGTTCCCCCACCAAAGCTG

256_232 TCGGTTGT AGAG TGGAAATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 364

NGSPLEx TCGCGAAAT GAGTG TCACAACAGGGGAGGCCTTGGTGAAAGCTGGGTGGAAA

256_233 TCGGTTGT AGTG ACCCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 365

NGSPLEx TCGCGAAAT CTGTG GTGGAGTCTAGAGGATCCACAGCTGGATAGATGCCCAG

256_234 TCGGTTGT ACAG CTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 366

NGSPLEx TCGCGAAAT CACTG AGGTAAAGGCCTGCAGCGATGAAACAGTTGTAGCTGACT

256_235 TCGGTTGT AGAG TGCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 367

NGSPLEx TCGCGAAAT CTCAG TCATTGATTGGTTGCCCGTCAAATCGGAATCTGATCTGCT

256_236 TCGGTTGT TCAG GGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 368

NGSPLEx TCGCGAAAT CTGTG GCTGAAACTTTCACAGGCTTCACAATCTTTTGCTTAGGTG

256_237 TCGGTTGT TGAG CTGCCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 369

NGSPLEx TCGCGAAAT GTGAC CACATAGAAGTCCAGGCCGTAGATACCAATGCTTGGTGG

256_238 TCGGTTGT AGTG TCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 370

NGSPLEx TCGCGAAAT CTGAC ACCATGCCCAGCACATCCTGCACATGCTGGCCCAGGTTG 256_239 TCGGTTGT TCAG GAGCCCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 371

NGSPLEx TCGCGAAAT GTCAG GGTGATGGTAGCCTTTCTGCCCAGCGCGTGCCACAGTG

256_240 TCGGTTGT ACTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 372

NGSPLEx TCGCGAAAT CACTG GCCGCATCCGCGTCAGATTCCCAAACTCGCGGCCCATTG

256_241 TCGGTTGT AGTG TGGCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 373

NGSPLEx TCGCGAAAT CTCAG TTGCTGTCACCAGCAACGTTGCCACGACGAACATCCTTG

256_242 TCGGTTGT AGAG ACAGACATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 374

NGSPLEx TCGCGAAAT CAGTC GCTGGTATAAGGTGGTCTGGTTGACTTCTGGTGTCCCCA

256_243 TCGGTTGT ACAG CGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 375

NGSPLEx TCGCGAAAT GAGAG CGGCATCCTCTCAGGAGGGCCGGTCCGGGTCTCAGCGC

256_244 TCGGTTGT AGTG GCCTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 376

NGSPLEx TCGCGAAAT CAGAC AGGTTAACCATGTGCCCGTCGATGTCCTTGGCGGAAAAC

256_245 TCGGTTGT AGAG TCGTGCATGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 377

NGSPLEx TCGCGAAAT GAGTC ACCACAAACTCTTCCACCAGCCAGCATGGCAAATTTGAG

256_246 TCGGTTGT TGTG GTGCTTGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 378

NGSPLEx TCGCGAAAT CTGAC TGGAGATTGCAGTGAGCTGAGATCACACCACTGGGCTCC

256_247 TCGGTTGT TCTG AGCCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 379

NGSPLEx TCGCGAAAT CAGTG GGCCAGTGGTCTTGGTGTGCTGGCCTCGGACACGAATG

256_248 TCGGTTGT TGTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 380

NGSPLEx TCGCGAAAT CACAG GCAGCTGGAGCATCTCCACCCTTGGTATTTCTGGTGTAA

256_249 TCGGTTGT ACTG TGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 381

NGSPLEx TCGCGAAAT CTCTC GTAGCTGGGGGTGCTGGGGTTCATTCTCGGCACGGCTG

256_250 TCGGTTGT AGTG CTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 382

NGSPLEx TCGCGAAAT GTCAC GCTGTAACCACACCGACGCGCGAGCTCTGCGCGGGCTT

256_251 TCGGTTGT AGTG CACTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 383

NGSPLEx TCGCGAAAT CTGTC TCCAGGTCGATCTCCAAGGACTGGACTGTACGTCTCAGC

256_252 TCGGTTGT AGAG TCTTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 384

NGSPLEx TCGCGAAAT GACAG TTAACCTACCACTG I I I I GTTTAGAGCGAACACAGTGTGG

256_253 TCGGTTGT TGTG TCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 385

NGSPLEx TCGCGAAAT GACAC TCTCCTCCAGGGTGGCTGTCACTGCCTGGTACTTCCATG

256_254 TCGGTTGT ACTG GTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 386

NGSPLEx TCGCGAAAT GAGTG CACGACAGCAAATAGCACGGGTCAGATGCCCTTGGCTGA

256_255 TCGGTTGT ACTG AAAGTGGTCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 387

NGSPLEx TCGCGAAAT GTCAC GGGACCAGCCGTCCTTATCAAAGTGCTCCCAGAAATTGG

256_256 TCGGTTGT TCAG TCGGTGCTCGCAGGCTCGGCA

SEQ ID NO: 131 SEQ ID NO: 388

Name Region 1 Region 2

Capture

ACAACCGAATTTCGCGAT TTTTTTTTTTVBiotin

Probe256 CWSWSWS

plex WS (SEQ ID NO: 389) Table 3 - List of sequences used for proof of concept experiment Fig. 6

Name Region 3 Region 4 Region 5

Precur TAGCGCCT CTCTCT TAGCGCCTGCGGCCTGTCTCTCTCTGUAAACGCATCGG sor GCGGCCT CTGU TCGAATTATCTCCTGCTAGGCACTCGCTGTGCCCTGGA

Oligo 1 GT CTATCGTAACCCATGCTGTTT/36-FAM-3'

SEQ ID NO: SEQ ID NO: SEQ ID NO: 392

390 391

Precur TAGCGCCT GTGTGA CTTGCGGAACACGAATCGACCACTGACACAATTCGTAAT sor GCGGCCT CTGU CTCATTGCAAGCGTTT/36-FAM-3'

Oligo 2 GT

SEQ ID NO: SEQ ID NO: SEQ ID NO: 394

390 393

Precur TAGCGCCT CAGAGA ATGCCCATTCAGCCTCACGTGGTGCTGATTTGGGGTGT sor GCGGCCT CTGU TT/36-FAM-3/

Oligo 3 GT

SEQ ID NO: SEQ ID NO: SEQ ID NO: 396

390 395

Name Region 1 Region 2

Captur

Probe2 CAGWSWS ACAGGCC

56plex WS (SEQ ID NO

TTaabbllee 44■ - List of sequences used for Fig. 10

Name/

6 7

Region

Precur GTGGATGATC ACACA

sor AACGC CGCATTTAGAGTGAAGTATCAATCGGAAATCGTGC

Oligo 4 AGCGACC/36-FAM-3'

SEQ ID NO: 398 SEQ ID NO: 399

Precur GTGGATGATC ACTGT UCAATCAACCAGATTAGGACTCGGTTCCCGTGAGA sor AACGC AATAGAAGTCCGTATAAACGTTCAACGGGGTC/36-

Oligo 5 FAM-3'

SEQ ID NO: 398 SEQ ID NO: 400

Precur GTGGATGATC AGTC! UCGCTTCCATACCGGGCGATGGACACAATTAAGAT sor AACGC CGCATTTAGAGT/36-FAM-3'

Oligo 6

SEQ ID NO: 398 SEQ ID NO: 401

Name/

1 4 2

Region

Captur GWSWSWST GCGTTGAT ATAGACTCG/iBiodT/TTTTTTT/iBiodT/TTTTTTT/3B e CATCCAC

Probe

SEQ ID NO: SEQ ID NO: 403

402

Name/

Region

s

Protect AGTCTAT GTGGATGA

or TCAACGC

SEQ ID NO:

398 All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

XI. References

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

SantaLucia Jr, J., & Hicks, D. (2004). The thermodynamics of DNA structural motifs. Annu. Rev. Biophys. Biomol. Struct., 33, 415-440.

Wu, L. R., Wang, J. S., Fang, J. Z., Evans, E. R., Pinto, A., Pekker, I., ... & Zhang, D. Y. (2015). Continuously tunable nucleic acid hybridization probes. Nature methods, 12(12), 1191-1196.

Zhang, D. Y., Chen, S. X., & Yin, P. (2012). Optimizing the specificity of nucleic acid hybridization. Nature chemistry, 4(3), 208-214.