WO2016079509A1

WO2016079509A1 - Methods for nucleic acid isolation

Info

Publication number: WO2016079509A1
Application number: PCT/GB2015/053500
Authority: WO
Inventors: Tobias William Barr Ost; Neil Matthew BELL; Christine Clark; Andreas Claas
Original assignee: Cambridge Epigenetix Limited
Priority date: 2014-11-18
Filing date: 2015-11-18
Publication date: 2016-05-26
Also published as: GB201420428D0

Abstract

This invention relates to improved methods and kits for identification of modified cytosines in a sample nucleotide sequence. Methods comprise treating the nucleic acid sample with bisulfite, adding a suspension of beads in polyethylene glycol without any additional salt such that the nucleic acids bind to the beads, thus enabling purification of the nucleic acid sample. These purification methods may be useful, for example in the analysis of genomic DNA and/or of RNA which has been bisulfite treated.

Description

Methods for Nucleic Acid Isolation

This invention relates to the detection of modified cytosine residues and, in particular, to the sequencing of nucleic acids that contain modified cytosine residues . The sequencing of nucleic acids containing modified cytosine residues requires the isolation of the nucleic acid sample from a solution containing bisulfite. An improved method of nucleic acid isolation involves the use of beads to bind bisulfite treated nucleic acids.

5-methylcytosine (5mC) is a well-studied epigenetic DNA mark that plays important roles in gene silencing and genome

stability, and is found enriched at CpG dinucleotides (2) . In metazoa, 5mC can be oxidised to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation (TET) family of enzymes {2, 3) . The overall levels of 5hmC are roughly 10-fold lower than those of 5mC and vary between tissues (4) . Relatively high quantities of 5hmC (~0.4% of all cytosines) are present in embryonic stem (ES) cells, where 5hmC has been suggested to have a role in the establishment and/or maintenance of pluripotency {2,3, 5-9) . 5hmC has been proposed as an intermediate in active DNA

demethylation, for example by deamination or via further oxidation of 5hmC to 5-formylcytosine (5fC) and 5- carboxycytosine (5cC) by the TET enzymes, followed by base excision repair involving thymine-DNA glycosylase (TDG) or failure to maintain the mark during replication (20). However, 5hmC may also constitute an epigenetic mark per se.

It is possible to detect and quantify the level of 5hmC present in total genomic DNA by analytical methods that include thin layer chromatography and tandem liquid chromatography-mass spectrometry {2, 11, 12) . Mapping the genomic locations of 5hmC has thus far been achieved by enrichment methods that have employed chemistry or antibodies for 5hmC-specific precipitation of DNA fragments that are then sequenced {6-8, 13-15) . These pull-down approaches have relatively poor resolution (10s to information that is likely to be subject to distributional biasing during the enrichment. Quantifiable single nucleotide sequencing of 5mC has been performed using bisulfite sequencing (BS-Seq) , which exploits the bisulfite-mediated deamination of cytosine to uracil for which the corresponding transformation of 5mC is much slower {16) . However, it has been recognized that both 5mC and 5hmC are very slow to deaminate in the bisulfite reaction and so these two bases cannot be discriminated {17, 18) . Two relatively new and elegant single molecule methods have shown promise in detecting 5mC and 5hmC at single nucleotide resolution. Single molecule real-time sequencing (SMRT) has been shown to detect derivatised 5hmC in genomic DNA {19) . However, enrichment of DNA fragments containing 5hmC is required, which leads to loss of quantitative information {19) . 5mC can be detected, albeit with lower accuracy, by SMRT {19) . Furthermore, SMRT has a relatively high rate of sequencing errors {20) , the peak calling of modifications is imprecise {19) and the platform has not yet sequenced a whole genome. Protein and solid-state nanopores can resolve 5mC from 5hmC and have the potential to sequence unamplified DNA molecules with further development {21, 22) .

The present inventors have devised an improved method for purifying nucleic acid samples having a high level of salt, for example bisulfite treated nucleic acid samples or nucleic acid samples from caesium chloride purification gradients. These methods are applicable to all sequencing platforms and may be useful, for example in the analysis of genomic DNA and/or of RNA.

Methods of purifying nucleic acid samples using solid phase binding is well known. A typical procedure involves capture onto a negatively charged bead surface (e.g carboxylate or silica) using a binding buffer containing polyethylene glycol (PEG) and a high level of salt. Typical salt concentrations of PEG/salt binding buffers is 1.25 M or greater. The problem identified by the inventors is that the treatment with bisulfite, or with caesium chloride purification, already exposes the nucleic aci samples to a high level of salt. Therefore the standard bead binding buffers, which also contain high levels of salt, mean that the salt level is too high once the solutions are combine The excess salt concentration causes a phase separation as the PEG becomes insoluble in the aqueous phase. The prior art process for the solid phase, bead based, purification of nucle acid samples therefore does not work effectively. The solution provided is to use a bead binding buffer with a lower level of salt than the prior art binding buffers.

The term salt is used herein to include mono, di and trivalent cations . The cations may be metals or ammonium ions . Typical cations include guanidinium, ammonium, sodium, potassium and magnesium. Salts include NaCl, KC1 and MgCl₂.

In WO 2009/070843, RNA treated with 1 M to 6 M of bisulfite was recovered by binding to a solid phase. The method employed to reduce the salt concentration in order to prevent interference with the binding step was to dilute the RNA sample to reduce salt concentration to below 1 M. This method is different from the present invention which uses an undiluted nucleic acid sample (with high salt content) and focusses on changing the salt content of the binding buffer itself. In addition, the present invention uses a binding buffer containing less than 1 M salt and polyethylene glycol. The binding conditions in WO 2009/070843 do not contain polyethylene glycol.

WO 2011/151428 describes conditions to bind nucleic acids to a solid phase such as beads. The binding conditions include the use of an amino containing binding ligand such as spermine or polyethylene imine and a salt concentration in the sample of less than 1 M. Binding is performed at pH of 3 to 7. Elution of the nucleic acids from the solid phase is achieved using elution buffers having a pH of about 7.5 to 9.0. In addition, the present invention uses a binding buffer containing less than 1 M salt and polyethylene glycol. The binding conditions in WO 2009/070843 do not require polyethylene glycol. The samples to be processed by the method disclosed in WO 2011/151428 do not include samples with high-salt content, samples with a salt concentration greater than 1 M or samples with bisulfite-treated DNA containing salt at a concentration greater than 1 M. In the absence of any samples already containing a high level of salt, it is not therefore apparent that a non-exemplified subset of the binding conditions disclosed in WO 2011/151428 will be essential to prevent interference with the binding step for high salt content samples. The present invention however specifically provides a method of purifying nucleic acid samples having a background salt concentration greater than 1 M. The present invention requires polyethylene glycol and does not require the use of binding ligands other than salt. The use of additional binding ligands such as amino containing compounds is

undesireable as the ligands can contaminate the sample.

U.S. Patent Application No. 2012/0245337 describes binding of nucleic acids to amino functionalised beads. The positively charged beads do not require PEG or salt in order to bind the nucleic acids. As for the method disclosed in WO 2011/151428, the samples to be processed by the method disclosed in U.S.

2012/0245337 do not specifically include samples with high-salt content, samples with a salt concentration greater than 1 M or samples with bisulfite-treated DNA containing salt at a

concentration greater than 1 M. It is therefore not apparent that the binding conditions disclosed in U.S. 2012/0245337 will be applicable for such high salt samples . The present invention however specifically provides a method of purifying nucleic acid samples having a background salt concentration greater than 1 M.

U.S. Patent No. 7,022,835 describes a method for binding nucleic acids to a solid phase in the presence of salt and polyethylene glycol. The preferred binding conditions use a salt concentration of 5 mM to 4 M and the polyethylene glycol is preferably used at a concentration of 5 to 40%. The salt concentration is therefore not limited to less than 1 M as in the present invention. In addition, the samples to be processed by the method disclosed in U.S. 7,022,835 do not specifically include samples with high-salt content, samples with a salt concentration greater than 1 M or samples with bisulfite-treated DNA containing salt at a concentration greater than 1 M. In the absence of any samples already containing a high level of salt, it is not therefore apparent that a non-exemplified subset of the binding conditions disclosed in U.S. 7,022,835 will be essential to prevent interference with the binding step for high salt content samples . The present invention however specifically provides a method of purifying nucleic acid samples having a background salt concentration greater than 1 M.

EP 1748072 describes conditions under which nucleic acids bind to paramagnetic particles. Unlike the current invention, salt concentrations of the nucleic acid sample are adjusted to between 0.1 M and 0.5 M before exposure to the binding solution or beads. In all three examples provided in EP 1748072, the bead binding solution contains 5 M salt in order to increase the salt concentration for binding to the beads, and the solution is then diluted by the addition of ethanol. Polyethylene glycol is not used for binding in any of the examples and it is merely suggested in the description that the controlled release of nucleic acid fragments based on size can be accomplished by adjusting the alcohol concentration, and/or the nature of the alcohol (ethanol vs. PEG, the molecular weight of the PEG, etc.) . There are no examples using a binding buffer comprising a salt concentration less than 1 M and polyethylene glycol. In addition, the samples to be purified with this method do not explicitly include samples with high-salt content, samples with a salt concentration greater than 1 M or samples with bisulfite- treated DNA containing salt at a concentration greater than 1 M. In the absence of any samples already containing a high level of salt, it is not therefore apparent that a non-exemplified subset of the binding conditions disclosed in EP 1748072 will be essential to prevent interference with the binding step for high salt content samples . The present invention however specifically provides a method of purifying nucleic acid samples having a background salt concentration greater than 1 M.

WO 99/58664 describes a method to isolate nucleic acids from a mixture using polyethylene glycol, salt and solid phase carriers such as beads. WO 99/58664 teaches that yields of bound DNA decrease if the salt concentration is less than about 0.5 M or greater than about 5.0 M. It is not specified that the binding buffer should contain less than 1 M salt. The example 1 of WO 99/58664 uses a salt concentration of 3.3 M for bead binding. The concentration of salt in the binding buffer is therefore not limited to less than 1 M as in the present invention. In addition, samples to be purified with the WO 99/58664 method do not explicitly include samples with high-salt content, samples with a salt concentration greater than 1 M or samples with bisulfite-treated DNA containing salt at a concentration greater than 1 M. In the absence of any samples already containing a high level of salt, it is not therefore apparent that a non- exemplified subset of the binding conditions disclosed in WO 99/58664 will be essential to prevent interference with the binding step for high salt content samples. The present

invention however specifically provides a method of purifying nucleic acid samples having a background salt concentration greater than 1 M.

WO 2010/115016 discloses a method to extract short nucleic acids from a nucleic acid composition containing a background of longer nucleic acids . Sample nucleic acids to be purified by the method include bisulfite-treated nucleic acids. The method uses a solid support such as beads to which nucleic acids may reversibly bind. The binding conditions may include a salt concentration in the range of about 0.25 M to about 5 M and may also include polyethylene glycol as a volume excluding agent. As stated in EP 1748072, the release of nucleic acid fragments based on size can be accomplished by adjusting the alcohol concentration, and/or the nature of the alcohol (ethanol vs. PEG, the molecular weight of the PEG, etc.) . As opposed to the current invention, the method disclosed in WO 2010/115016 uses binding conditions with 1.4 M guanidine thiocyanate. The salt concentration is therefore not limited to less than 1 M as in the present invention. In addition, the samples to be purified with the WO 2010/115016 method do not explicitly include samples with high-salt content, samples with a salt concentration greater than 1 M or samples with bisulfite-treated DNA

containing salt at a concentration greater than 1 M. The present invention specifically provides a method of purifying nucleic acid samples with a background salt concentration greater than 1 M, whereby the binding solution has a salt concentration less than 1 M.

An aspect of the invention provides a method of purifying a nucleic acid sample comprising

(i) providing a population of polynucleotides which comprise the nucleic acid sample and greater than 1 M background salt,

(ii) mixing polynucleotides with a suspension of beads in a binding buffer containing polyethylene glycol and less than 1 M salt ;

thereby binding the polynucleotides to the beads .

(ii) providing a suspension of beads in a buffer containing polyethylene glycol and less than 1 M salt; and

(iii) mixing the population of polynucleotides with the

suspension of beads, thereby binding the population of

polynucleotides to the beads . The beads to which the polynucleotides are added are suspended in a solution having a total salt concentration of less than 1 M. The salt may be made up of a mixture of different ions, or may be a single cation and a single anion. The term salt is used herein to include mono, di and trivalent cations. The cations may be metals or ammonium ions. Typical cations include

guanidinium, ammonium, sodium, potassium and magnesium. Salts include NaCl, KC1 and MgCl₂. The beads to which the

polynucleotides are added contain a solution having a NaCl concentration of less than 1 M.

The background salt may come from a bisulfite treatment step. The presence of the bisulfite counterions means that a lower level of salt, or indeed no salt at all, is required to cause binding to beads. Thus rather than the prior art PEG/Salt buffer, the binding buffer can contain a lower level of salt due to the presence of the counterions from the bisulfite.

The bisulfite solution may be provided in the form of sodium bisulfite, potassium bisulfite or ammonium bisulfite. Where the term bisulfite is used, the bisulfite may be obtained from any source, including metabisulfite . Thus the bisulfite may be provided by ammonium, sodium or potassium bisulfite or

metabisulfite . The presence of the ammonium, sodium or potassium ions means that a lower level of salt is required in the bead binding buffer.

The beads may be magnetic or paramagnetic. The beads can have a negative charge at the pH of the binding buffer . The beads can be carboxylate beads . The beads can be polymeric. The beads can be silica beads .

The polyethylene glycol may be present at 5% to 50% by volume of the binding buffer. The polyethylene glycol may be present at 7% to 25% by volume of the binding buffer. The polyethylene glycol may be present at 10% to 20% by volume of the binding buffer. The reduced salt binding buffer may contain less than 0.5 M salt. The reduced salt binding buffer may contain less than 0.1 M salt. The binding buffer may contain no added salt.

The reduced salt binding buffer may have a pH of between 7.0 and 9.0. The pH may be around 8.0. The buffer may contain a low ionic strength of a suitable buffering agent, for example tris (hydroxymethyl ) aminomethane (tris) . The concentration of the buffer may be for example 1-20 mM. The concentration of the buffer may be 5-10 mM.

The reduced salt binding buffer does not require any further binding ligands . The binding buffer does not contain any compounds containing amino groups such as spermine or

polyethylene imine . The binding to the nucleic acid sample containing a high salt concentration to the beads can be carried out in a buffer consisting PEG and a low ionic strength buffer to control the pH.

The polyethylene glycol may be any molecular size that remains soluble in water. The polyethylene glycol may be of molecular size 4000-35000. The polyethylene glycol may be of molecular size 6000-8000. The polyethylene glycol may be PEG 6000-PEG 8000.

Once the polynucleotides are bound to the beads, the solution may be removed (or the beads removed from the solution) . The separated beads may be washed to remove any loosely adhered matter. The washing may be carried out with a solution

containing an organic solvent. The solution may be 70% ethanol or greater than 70% ethanol. The solution may be 70%

acetonitrile or greater than 70% acetonitrile .

The beads may be exposed to conditions where the polynucleotides are desulfonated whilst remaining bound to the beads . The beads may be exposed to a buffer having a pH greater than 10. The beads may be treated with a buffer containing sodium hydroxide in aqueous ethanol. The buffer may contain 0.3 M hydroxide in 70% ethanol.

In order to further analyse the polynucleotides, the

polynucleotides can be removed from the beads. The removal is typically done using a low salt buffer, for example a buffer having less than 0.1 M salt, or with a high pH buffer, for example a buffer with a pH greater than 8.0.

Also disclosed are kits for the purification of nucleic acid samples. In addition to other optional components, the kits may contain;

(i) a bisulfite reagent;

(ii) beads; and

(iii) a binding buffer containing polyethylene glycol and less than 1 M salt.

The beads and binding buffer may be supplied separately, or the beads may be suspended in the binding buffer.

Disclosed is a kit for use in a method of purifying a nucleic acid sample comprising;

(i) a bisulfite reagent;

(ii) beads suspended in a buffer containing polyethylene glycol and less than 1 M salt.

The kit may contain a binding buffer having less than 0.1 M salt. The salt may be sodium chloride.

Upon isolation, the polynucleotides may be analysed, for example by sequencing. The sequencing may be compared with aliquots of the same sample which have not undergone bisulfite treatment. Samples which have not been exposed to bisulfite can be isolated into beads using the standard PEG/salt binding buffer. A first aliquot of the sample may be bisulfite treated, and a second aliquot may not be bisulfite treated. Cytosine residues may be present at one or more positions in the sample nucleic acid sequence. The residues at these one or more positions in the first and second nucleotide sequences may be identified. A modified cytosine at a position in the sample nucleotide sequence may be identified from a combination of residues identified in the first and second nucleotide sequences

respectively (i.e. C and C or U and C) at that position.

In some embodiments of the invention, a portion of

polynucleotides from the population may be reduced. The cytosine modifications which are indicated by different combinations are shown in table 1. In particular examples, unmodified C residues become U residues upon bisulfite treatment. 5-Formyl residues also become U residues upon bisulfite treatment. However if the formyl group is reduced to hydroxymethyl prior to bisulfite treatment, the base remains as C when treated with bisulfite. Thus the reduction step allows the differentiation of C and 5- formyl C which can not be distinguished by bisulfite treatment alone .

In some embodiments of the invention, a portion of

polynucleotides from the population may be oxidised. For example, 5-hydroxymethylcytosine residues in the first portion of polynucleotides may be converted into 5-formylcytosine (5fC) by oxidation and the portion of polynucleotides then treated with bisulfite. The oxidation in addition to the reduction allows differentiation between methylcytosine and

hydroxymethylcytosine .

A method of identifying a modified cytosine residue in a sample nucleotide sequence may comprise;

(i) providing a population of polynucleotides which comprise the sample nucleotide sequence, (ii) treating a first portion of said population with bisulfite,

(iii) mixing the bisulfite treated polynucleotides with suspension of beads in a binding buffer containing polyethyle glycol and less than 1 M salt,

(iv) separating the beads from the solution,

(v) washing the beads,

(vi) desulfonating the polynucleotides,

(vii) removing the polynucleotides from the beads,

(viii) taking a second portion of said population which has not undergone bisulfite treatment and sequencing the polynucleotides in the first and second portions of the

population to produce first and second nucleotide sequences, respectively and;

(ix) identifying the differences between the first and second nucleotide sequences, thereby identifying one or more modified cytosine residue in the sample nucleotide sequence.

The identification of a residue at a position in the first and second portions as cytosine is indicative that the cytosine residue in the sample nucleotide sequence is 5-methylcytosine or 5-hydroxymethylcytosine . 5-Methylcytosine and 5- hydroxymethylcytosine are not deaminated by the bisulfite treatment step.

The identification of a residue at a position in the first portion as uracil is indicative that the cytosine residue in the sample nucleotide sequence is unmodified cytosine or 5-formyl cytosine. Unmodified cytosine and 5-formyl cytosine are both deaminated by the bisulfite treatment step.

If desired, reduction and/or oxidation steps can be carried out on further portions of the sample. Thus the four states C, 5mC, 5hmC and 5fC can be distinguished by comparing the same

locations across the separate sequencing reactions on the different portions of the sample. The portions of the polynucleotide population may be treated with bisulfite and/or sequenced simultaneously or sequentially. The reducing step does not have to be performed prior to the oxidation step. A method indicated by roman numerals merely shows that the steps have to be carried out separately, not chronologically, unless explicitly stated or naturally inherent in the method.

In embodiments in which a portion is reduced, oxidation

treatment may not be required to identity or distinguish a modified cytosine residue in the sample nucleotide sequence. For example, Table 1 shows that reduction and bisulfite treatment of the first portion of the polynucleotide population is sufficient to identify 5-formylcytosine in the sample nucleotide sequence. A method of identifying 5-formylcytosine in a sample nucleotide sequence or distinguishing 5-formylcytosine from cytosine (C) , 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in a sample nucleotide sequence may comprise;

(i) providing a population of polynucleotides which comprise the sample nucleotide sequence,

(ii) reducing said population by adding an alkaline borohydride solution,

(iii) treating the reduced portion of said population and a second portion of said population with bisulfite,

(iv) mixing each bisulfite treated polynucleotides with a suspension of beads in a binding buffer containing polyethylene glycol and less than 1 M salt,

(v) separating the beads from the solution,

(vi) washing the beads,

(vii) desulfonating the polynucleotides,

(viii) removing the polynucleotides from the beads,

(ix) taking a third portion of said population which has not undergone bisulfite treatment and sequencing the

polynucleotides in the first, second and third portions of the population to produce first, second and third nucleotide sequences, respectively and; (x) identifying the differences between the first, second and third nucleotide sequences .

In order to differentiate between 5mC and 5hmC, an optional oxidation step may be introduced {23-25) . A summary of the cytosine modifications at a position in the sample nucleotide sequence which are indicated by specific combinations of cytosine and uracil at the position in the first, second and third nucleotide sequences is shown in Table 1. The four structures C, 5mC, 5hmC and 5fC are shown in Table 2.

The sample nucleotide sequence may be already known or it may b determined. The sample nucleotide sequence is the sequence of untreated polynucleotides in the population i.e. polynucleotide which have not been oxidised, reduced or bisulfite treated. In the sample nucleotide sequence, modified cytosines are not distinguished from cytosine. 5-Methylcytosine, 5-formylcytosine and 5-hydroxymethylcytosine are all indicated to be or

identified as cytosine residues in the sample nucleotide sequence .

The sequence of the polynucleotides may be determined by any appropriate sequencing technique.

The positions of one or more cytosine residues in the sample nucleotide sequence may be determined. This may be done by standard sequence analysis. Since modified cytosines are not distinguished from cytosine, cytosine residues in the sample nucleotide sequence may be cytosine, 5-methylcytosine, 5- formylcytosine or 5-hydroxymethylcytosine.

The first and second nucleotide sequences and, optionally the third nucleotide sequence, may be compared to the sample nucleotide sequence. For example, the residues at positions in the first and second sequences and, optionally the third nucleotide sequence, corresponding to the one or more cytosine residues in the sample nucleotide sequence may be identified.

The modification of a cytosine residue in the sample nucleotide sequence may be determined from the identity of the nucleotides at the corresponding positions in the first and second

nucleotide sequences and, optionally the third nucleotide sequence .

The polynucleotides in the population all contain the same sample nucleotide sequence i.e. the sample nucleotide sequence is identical in all of the polynucleotides in the population.

The effect of different treatments on cytosine residues within the sample nucleotide sequence can then be determined, as described herein.

The sample nucleotide sequence may be a genomic sequence. For example, the sequence may comprise all or part of the sequence of a gene, including exons, introns or upstream or downstream regulatory elements, or the sequence may comprise genomic sequence that is not associated with a gene. In some

embodiments, the sample nucleotide sequence may comprise one or more CpG islands .

The sample polynucleotides may be in single stranded or double stranded form. If the polynucleotides are in single stranded form, the concentration of borohydride may be lower that the concentration required for double stranded polynucleotides. The sample polynucleotides may therefore be denatured before the borohydride is added. The denaturation may take the form of heat or alkali. The method may include the step of treating the sample with alkali before the borohydride is added.

The final concentration of borohydride used to reduce the nucleic acid sample may be in the range of 10 to 500 mM. The concentration may be less than 0.2 M. Prior art reduction conditions carried out on double stranded DNA use solid

borohydride added to the solution at a higher concentration than 0.2 M. The concentration of borohydride may be 10 to 200 mM.

Suitable polynucleotides include DNA, preferably genomic DNA, and/or RNA, such as genomic RNA (e.g. mammalian, plant or viral genomic RNA) , mRNA, tRNA, rRNA and non-coding RNA.

The polynucleotides comprising the sample nucleotide sequence may be obtained or isolated from a sample of cells, for example, mammalian cells, preferably human cells.

Suitable samples include isolated cells and tissue samples, such as biopsies.

Modified cytosine residues including 5hmC and 5fC have been detected in a range of cell types including embryonic stem cells (ESCS) and neural cells {2 _r 3, 11, 37, 38) .

Suitable cells include somatic and germ-line cells.

Suitable cells may be at any stage of development, including fully or partially differentiated cells or non-differentiated or pluripotent cells, including stem cells, such as adult or somatic stem cells, foetal stem cells or embryonic stem cells.

Suitable cells also include induced pluripotent stem cells (iPSCs), which may be derived from any type of somatic cell in accordance with standard techniques.

For example, polynucleotides comprising the sample nucleotide sequence may be obtained or isolated from neural cells,

including neurons and glial cells, contractile muscle cells, smooth muscle cells, liver cells, hormone synthesising cells, sebaceous cells, pancreatic islet cells, adrenal cortex cells, fibroblasts, keratinocytes , endothelial and urothelial cells, osteocytes, and chondrocytes.

Suitable cells include disease-associated cells, for example cancer cells, such as carcinoma, sarcoma, lymphoma, blastoma or germ line tumour cells.

Suitable cells include cells with the genotype of a genetic disorder such as Huntington's disease, cystic fibrosis, sickle cell disease, phenylketonuria, Down syndrome or Marfan syndrome.

Methods of extracting and isolating genomic DNA and RNA from samples of cells are well-known in the art. For example, genomic DNA or RNA may be isolated using any convenient isolation technique, such as phenol/chloroform extraction and alcohol precipitation, caesium chloride density gradient centrifugation, solid-phase anion-exchange chromatography and silica gel-based techni.Ques .

In some embodiments, whole genomic DNA and/or RNA isolated from cells may be used directly as a population of polynucleotides as described herein after isolation. In other embodiments, the isolated genomic DNA and/or RNA may be subjected to further preparation steps .

The genomic DNA and/or RNA may be fragmented, for example by sonication, shearing or endonuclease digestion, to produce genomic DNA fragments. A fraction of the genomic DNA and/or RNA may be used as described herein. Suitable fractions of genomic DNA and/or RNA may be based on size or other criteria. In some embodiments, a fraction of genomic DNA and/or RNA fragments which is enriched for CpG islands (CGIs) may be used as

described herein.

The genomic DNA and/or RNA may be denatured, for example by heating or treatment with a denaturing agent . Suitable methods for the denaturation of genomic DNA and RNA are well known in the art .

In some embodiments, the genomic DNA and/or RNA may be adapted for sequencing before oxidation or reduction and bisulfite treatment, or bisulfite treatment alone. The nature of the adaptations depends on the sequencing method that is to be employed. For example, for some sequencing methods, primers may be ligated to the free ends of the genomic DNA and/or RNA fragments following fragmentation. Suitable primers may contain 5mC to prevent the primer sequences from altering during oxidation or reduction and bisulfite treatment, or bisulfite treatment alone, as described herein. In other embodiments, the genomic DNA and/or RNA may be adapted for sequencing after oxidation, reduction and/or bisulfite treatment, as described herein .

Following fractionation, denaturation, adaptation and/or other preparation steps, the genomic DNA and/or RNA may be purified by any convenient technique .

Following preparation, the population of polynucleotides may be provided in a suitable form for further treatment as described herein. For example, the population of polynucleotides may be in aqueous solution in the absence of buffers before treatment as described herein.

Polynucleotides for use as described herein may be single or double-stranded .

The population of polynucleotides may be divided into two, three, four or more separate portions, each of which contains polynucleotides comprising the sample nucleotide sequence. These portions may be independently treated and sequenced as described herein . The first portion of the population of polynucleotides

comprising the sample nucleotide sequence may be reduced.

Reduction converts any 5-formylcytosine in the sample nucleotide sequence to 5-hydroxymethylcytosine . The reduction may be carried out by adding an alkaline borohydride solution.

The use of a stabilised solution of borohydride allows improved kits for better control of the amount of borohydride added to the reaction. Borohydride solutions can be stabilised by a high pH. Thus the use of alkaline solution of borohydride gives control over the amount of active borohydride added to the nucleic acid sample. Prior art conditions of making borohydride solutions immediately prior to use means that the amount of active borohydride in solution depends of the purity and source of the borohydride, the level of decomposition of the solid prior to making the solution, the composition and pH of the buffer used to make the solution, and how long the solution is kept before use.

The need to make up a solution from a reactive powder

immediately prior to use does not allow the reducing agent to be supplied in a reliable way. One improvement described herein is therefore improved method and kits whereby a stabilised

borohydride solution is provided, thus allowing commercial distribution of improved kits, and more reliable methods whereby the reducing conditions can be controlled and reliably repeated.

The alkaline borohydride solution can be a metal borohydride. The borohydride can be lithium, sodium or potassium. The borohydride can be NaBH₄. Suitable reducing agents include

NaBH₄ , NaCNBH₄ and LiBH₄.

The alkaline borohydride can be supplied at a pH greater than 10.0. The solution can be sodium borohydride at pH greater than 10.0. The alkaline conditions can be ; >rovide by a solution containing hydroxide . The hydroxide can be lithium, sodium or potassium, The hydroxide can be present at a concentration of greater than 5 Moles/L. The hydroxide can be present at a concentration of greater than 10 Moles/L.

The optional oxidising agent is any agent suitable for

generating an aldehyde from an alcohol. The oxidising agent or the conditions employed in the oxidation step may be selected s that any 5-hydroxymethylcytosine is selectively oxidised. Thus, substantially no other functionality in the polynucleotide is oxidised in the oxidation step. The oxidising step therefore does not result in the reaction of any thymine or

5-methylcytosine residues, where such are present. The agent or conditions are selected to minimise or prevent any degradation of the polynucleotide.

The use of an oxidising agent may result in the formation of some corresponding 5-carboxycytosine product. The formation of this product does not negatively impact on the methods of identification described herein. Under the bisulfite reaction conditions that are used to convert 5-formylcytosine to uracil, 5-carboxycytosine is observed to convert to uracil also. It is understood that a reference to 5-formylcytosine that is obtaine by oxidation of 5-hydroxymethylcytosine may be a reference to a product also comprising 5-carboxycytosine that is also obtained by that oxidization.

The oxidising agent may be a non-enzymatic oxidising agent, for example, an organic or inorganic chemical compound.

Suitable oxidising agents are well known in the art and include metal oxides, such as KRu0₄, Mn0₂ and KMn0₄. Particularly useful oxidising agents are those that may be used in aqueous

conditions, as such are most convenient for the handling of the polynucleotide. However, oxidising agents that are suitable for use in organic solvents may also be employed where practicable.

In some embodiments, the oxidising agent may comprise a

perruthenate anion (Ru0₄ ^~) . Suitable perruthenate oxidising agents include organic and inorganic perruthenate salts, such as potassium perruthenate (KRu04) and other metal perruthenates; tetraal kylammonium perruthenates, such as tetrapropylammonium perruthenate (TPAP) and tetrabutylammonium perruthenate (TBAP) ; polymer-supported perruthenate (PSP) and tetraphenylphosphonium ruthenate .

Advantageously, the reducing and/or oxidising agent or the reducing conditions may also preserve the polynucleotide in a denatured state.

Following treatment with the reducing agent, the polynucleotides in the first portion may be purified.

Purification may be performed using any convenient nucleic acid purification technique. Suitable nucleic acid purification techniques include spin-column chromatography.

The polynucleotide may be subjected to further, repeat reducing steps. Such steps are undertaken to maximise the conversion of 5-formylcytosine to 5-hydroxymethylcytosine . This may be necessary where a polynucleotide has sufficient secondary structure that is capable of re-annealing. Any annealed portions of the polynucleotide may limit or prevent access of the reducing agent to that portion of the structure, which has the effect of protecting 5-formylcytosine from reduction.

In some embodiments, the first portion of the population of polynucleotides may for example be subjected to multiple cycles of treatment with the reducing agent followed by purification. For example, one, two, three or more than three cycles may be performed .

Following oxidation and reduction, the portions of the

population are treated with bisulfite. The bisulfite may be generated from a solution of metabisulfite . A portion of the population which has not been oxidised or reduced is also treated with bisulfite. Bisulfite treatment converts both cytosine and 5-formylcytosine residues in a polynucleotide into uracil. A portion of the population may be treated with

bisulfite by incubation with bisulfite ions (HS0₃ ^~) .

The use of bisulfite ions (HS0₃ ^~) to convert unmethylated cytosines in nucleic acids into uracil is standard in the art and suitable reagents and conditions are well known to the skilled person {39-42). Numerous suitable protocols and reagents are also commercially available (for example, EpiTect™, Qiagen NL; EZ DNA Methylation™, Zymo Research Corp CA; CpGenome Turbo Bisulfite Modification Kit, Millipore or Truemethyl™, Cambridge Epigenetix) .

A feature of the methods described herein is the conversion of unmethylated cytosine to uracil. This reaction is typically achieved through the use of bisulfite. However, in general aspects of the invention, any reagent or reaction conditions may be used to effect the conversion of cytosine to uracil. Such reagents and conditions are selected such that little or no 5- methylcytosine reacts, and more specifically such that little or no 5-methylcytosine reacts to form uracil. The reagent, or optionally a further reagent, may also effect the conversion of 5-formylcytosine or 5-carboxycytosine to cytosine or uracil.

Following the incubation, the portions of polynucleotides may be immobilised, washed, desulfonated, eluted and/or otherwise treated as required. The bisulfite treatment typically introduces significant level of salt into the reaction mixture. Typical bisulfite treatment conditions involve concentrations of bisulfite of greater than M, often 3 M or higher. Thus after bisulfite treatment, the nucleic acid sample is in a solution containing molar levels o cations, usually metal cations where sodium or potassium bisulfite are used.

Many nucleic acid purification procedures expose a nucleic acid sample to a suspension of beads in a binding buffer having PEG and a high level of salt, for example greater than 1.25 M NaCl . If such high salt solutions as those following bisulfite treatment are treated with further salt, then a binding buffer containing PEG becomes unstable and the PEG phase separates. Thus if the concentration of salt in the binding buffer is too high, rather than causing the nucleic acids to bind to the beads, the salt causes the PEG to separate from the solution, and nothing is bound to the beads at all. Thus is it important not to add excess salt to the binding buffer. Standard protocols for solid phase isolation of nucleic acids involve the addition of mixtures of PEG and salt to capture nucleic acids onto beads. Such processes fail where the sample already contains high level of salt, as the nucleic acids fail to be captured by the beads. Thus the inventors have solved the problem by using a process where a low level of salt (or indeed no salt at all) is added to the bead binding buffer. Thus bisulfite treated samples can be purified directly onto solid supports just by using the solid support in a PEG buffer. There is no need for the pre-dilution of the sample in order to lower the salt concentration.

The solid phase bound nucleic acids can be separated from the solution. If the beads are magnetic, the separation can be carried out using a magnet. The solution is no longer requirec and can be discarded. The beads can be washed to remove any loosely adhered material. The washing can be carried out with solution containing organic solvent. The solution can contain aqueous ethanol, for example 70% ethanol. The solution can contain aqueous acetonitrile, for example 70% acetonitrile .

The beads may be exposed to conditions where the polynucleotides are desulfonated whilst remaining bound to the beads. The beads may be exposed to a buffer having a pH greater than 10. The beads may be treated with a buffer containing sodium hydroxide in aqueous ethanol. The buffer may contain 0.3 M hydroxide in 70% ethanol.

Following washing, the beads can be treated with an elution buffer to release the bound material. The elution buffer can be low in salt, for example less than 0.1 M or have a high pH, for example >8.0. The buffer can be a tris based buffer. The tris buffer can contain no additional salt.

In some embodiments, the first, second and third portions of polynucleotides from the population may be amplified following treatment as described above. This may facilitate further manipulation and/or sequencing. Sequence alterations in the first, second and third portions of polynucleotides are

preserved following the amplification. Suitable polynucleotide amplification techniques are well known in the art and include PCR. The presence of a uracil (U) residue at a position in the first, second and/or third portions of polynucleotide may be indicated or identified by the presence of a thymine (T) residue at that position in the corresponding amplified polynucleotide.

As described above, polynucleotides may be adapted before or after oxidation, reduction and/or bisulfite treatment to be compatible with a sequencing technique or platform. The nature of the adaptation will depend on the sequencing technique or platform. For example, for Solexa-Illumina sequencing, the treated polynucleotides may be fragmented, for example by sonication or restriction endonuclease treatment, the free ends of the polynucleotides repaired as required, and primers ligated onto the ends. If the sample is exposed to bisulfite and desulfonation, the sample may end up as short single stranded fragments. The sample may be adapted using a single stranded sample preparation technique. Suitable single stranded sample preparation techniques include, for example, the use of template independent polymerases to join two oligonucleotides (26) .

Polynucleotides may be sequenced using any convenient low or high throughput sequencing technique or platform, including Sanger sequencing {43), Solexa-Illumina sequencing {44),

Ligation-based sequencing (SOLiD™) {45), pyrosequencing {46); strobe sequencing (SMRT™) {47, 48); and semiconductor array sequencing (Ion Torrent™) {49) .

Suitable protocols, reagents and apparatus for polynucleotide sequencing are well known in the art and are available

commercially.

The residues at positions in the first, second and/or third nucleotide sequences which correspond to cytosine or uracil may be identified.

The modification of a cytosine residue at a position in the sample nucleotide sequence may be determined from the identity of the residues at the corresponding positions in the first, second and, optionally, third nucleotide sequences, as described above .

The extent or amount of cytos ine modification in the sample nucleotide sequence may be determined. For example, the

proportion or amount of 5-hydroxymethylcytosine and/or

5-methylcytosine in the sample nucleotide sequence compared unmodified cytosine may be determined .

Polynucleotides as described herein, for example the population of polynucleotides or 1, 2, 3, or all 4 of the first, second, third and fourth portions of the population, may be immobilised on a solid support.

A solid support is an insoluble, non-gelatinous body which presents a surface on which the polynucleotides can be

immobilised. Examples of suitable supports include glass slides, microwells, membranes, or microbeads . The support may be in particulate or solid form, including for example a plate, a test tube, bead, a ball, filter, fabric, polymer or a membrane.

Polynucleotides may, for example, be fixed to an inert polymer, a 96-well plate, other device, apparatus or material which is used in a nucleic acid sequencing or other investigative context. The immobilisation of polynucleotides to the surface of solid supports is well-known in the art. In some embodiments, the solid support itself may be immobilised. For example, microbeads may be immobilised on a second solid surface.

In some embodiments, the first, second, third and/or fourth portions of the population of polynucleotides may be amplified before sequencing. Preferably, the portions of polynucleotide are amplified following the treatment with bisulfite.

Suitable methods for the amplification of polynucleotides are well known in the art.

Following amplification, the amplified portions of the

population of polynucleotides may be sequenced.

Nucleotide sequences may be compared and the residues at positions in the first, second and/or third nucleotide sequences which correspond to cytosine in the sample nucleotide sequence may be identified, using computer-based sequence analysis.

Nucleotide sequences, such as CpG islands, with cytosine modification greater than a threshoId value may be identified, For example, one or more nucleotide sequences in which greater than 1%, greater than 2%, greater than 3%, greater than 4% or greater than 5% of cytosines are hydroxymethylated may be identified .

Computer-based sequence analysis may be performed using any convenient computer system and software. A typical computer system comprises a central processing unit (CPU) , input means, output means and data storage means (such as RAM) . A monitor or other image display is preferably provided. The computer system may be operably linked to a DNA and/or RNA sequencer.

For example, a computer system may comprise a processor adapted to identify modified cytosines in a sample nucleotide sequence by comparison with first, second and/or third nucleotide sequences as described herein. For example the processor may be adapted;

(a) identify the positions of cytosine residues in the sample nucleotide sequence,

(b) identify the residues in the first, second and/or third nucleotide sequences at the positions of cytosine residue in the sample nucleotide sequence,

(c) determine from the identities of said residues the presence or absence of modification of the cytosine residue at the positions in the sample nucleotide sequence.

The sample nucleotide sequence and the first second and third nucleotide sequences may be entered into the processor

automatically from the DNA and/or RNA sequencer. The sequences may be displayed, for example on a monitor.

The computer system may further comprise a memory device for storing data. Nucleotide sequences such as genomic sequences, and the positions of 5fC, 5hmC and other modified cytosine residues may be stored on another or the same memory device, and/or may be sent to an output device or displayed on a monitor. This may facilitate the mapping of modified cytosines, such as 5hmC and 5fC, in genomic DNA.

The identification and mapping of cytosine modifications, such as 5fC and 5hmC, in the genome may be useful in the study of neural development and function, and cell differentiation, division and proliferation, as well as the prognosis and diagnosis of diseases, such as cancer.

The identification and/or mapping of modified cytosines such as 5fC and 5hmC, using the methods described herein may therefore be useful in disease.

Another aspect of the invention provides a kit for the

purification of nucleic acid samples. In addition to other optional components, the kits may contain;

(i) a bisulfite reagent;

(ii) beads; and

(iii) a binding buffer containing polyethylene glycol and less than 1 M salt.

The beads and binding buffer may be supplied separately, or the beads may be suspended in the binding buffer. The beads may be magnetic or paramagnetic. The beads can have a negative charge at the pH of the binding buffer. The beads can be carboxylate beads. The beads can be polymeric. The beads can be silica beads .

The bisulfite reagent may be bisulfite or metabisulfite . The bisulfite reagent may be a sodium, potassium or ammonium salt.

The polyethylene glycol may be present at 5% to 50% by volume o the binding buffer. The polyethylene glycol may be present at 7 to 25% by volume of the binding buffer. The polyethylene glycol may be present at 10% to 20% by volume of the binding buffer. The reduced salt binding buffer may contain less than 0.5 M salt. The reduced salt binding buffer may contain less than 0.1 M salt. The binding buffer may contain no added salt. The polyethylene glycol may be any molecular size that remains soluble in water. The polyethylene glycol may be of molecular size 4000-35000. The polyethylene glycol may be of molecular size 6000-8000. The polyethylene glycol may be PEG 6000-PEG 8000.

The kit may further contain an eluting buffer, for example a buffer having less than 0.1 M salt or a pH greater than 8.0.

The kit may comprise a kit for use in a method of identifying a 5-formylcytosine residue comprising;

(i) an alkaline borohydride solution,

(ii) a bisulfite reagent;

(iii) beads; and

(iv) a binding buffer containing polyethylene glycol and less than 1 M salt.

The alkaline borohydride solution can be a metal borohydride. The borohydride can be lithium, sodium or potassium. The borohydride can be NaBH₄. Suitable reducing agents include NaBH₄ , NaCNBH₄ and LiBH₄.

The alkaline borohydride can be supplied at a pH greater than 10.0. The alkaline borohydride can be supplied at a pH greater than 14.0. The solution can be sodium borohydride at pH greater than 10.0. The solution can be sodium borohydride at pH greater than 14.0. The borohydride can be present in the range of 1-30 weight % of the solution. The borohydride can be present in the range of 10-20 weight % of the solution.

The alkaline conditions can be provided by a solution containing hydroxide. The hydroxide can be lithium, sodium or potassium.

The hydroxide can be present at a concentration of greater than 1 Moles/L. The hydroxide can be present at a concentration of greater than 5 Moles/L. The hydroxide can be present at a concentration of greater than 10 Moles/L.

A kit may further comprise a population of control

polynucleotides comprising one or more modified cytosine residues, for example cytosine (C) , 5-methylcytosine (5mC), hydroxymethylcytosine (5hmC) or 5-formylcytosine (5fC) . In embodiments, the population of control polynucleotides may divided into one or more portions, each portion comprising different modified cytosine residue.

The kit may include instructions for use in a method of

identifying a modified cytosine residue as described above.

A kit may include one or more other reagents required for the method, such as buffer solutions, sequencing and other reagents, A kit for use in identifying modified cytosines may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself, including DNA and/or RNA isolation and purification reagents, and sample handling containers (such components generally being sterile) .

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety for all purposes .

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and

definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments that are described.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below. Table 1 shows sequencing outcomes for cytosine and modified cytosines subjected to various treatments.

Table 2 shows the structures of cytosine (la), 5-methylcytosine (5mC; lb), 5-hydroxymethylcytosine (5hmC; lc) and 5- formylcytosine (5fC; Id)

Figure 1: Post Bisulfite ssDNA Recovery Purification Method Comparison lkb fragmented Salmon Sperm Genomic DNA (Invitrogen) was bisulfite converted and purified direct from the bisulfite reaction using either an Amicon 30kDa ultracentrifuge filtration device or with magnetic beads using the salt-free BB2 buffer method prepared with either 36% PEG-4000 (BB2-4000) or 50% PEG- 10000 (BB2-10000) . The input mass to the bisulfite conversion was either 1000 or 100 ng . The recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit. Recoveries are shown in Figure 1, bars are labeled with the % recovery of ssDNA.

Figure 2: Post Bisulfite ssDNA Recovery Input Mass Titration (50 kb)

50kb Promega Human Genomic DNA was bisulfite converted and purified direct from the bisulfite reaction on magnetic beads using the salt-free BB2 buffer method. The input mass was titrated across the range 1000 - 250 ng and the recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit. Recoveries are shown in Figure 2, bars are labeled with the average % recovery (n=2), error bars show the standard deviation from the mean.

Figure 3: Post Bisulfite ssDNA Recovery Input Mass Titration (400bp)

400 bp fragmented Lambda Genomic DNA was bisulfite and

oxidative-bisulfite converted and purified direct from the bisulfite reaction on magnetic beads using the salt-free BB2 buffer method. The input mass was titrated across the range 800 - 50 ng and the recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit. Recoveries are shown in Figure 3, bars are labeled with the average % recovery (n=2), error bars show the standard deviation from the mean. Bars labeled oxBS+BS report the composite recovery values from oxidative bisulfite and bisulfite converted samples .

Figure 4 : Post Bisulfite ssDNA Recovery Different gDNA Types Input DNA was either 50kb human blood leukocyte gDNA (AMSbio) or

50 kb human cerebellum gDNA (AMSbio) . 6 replicate experiments were performed with each genomic DNA type. The total input mass for each sample was 1 g, which was subsequently split into two separate 500 ng aliquots and each aliquot individually subjected to either oxidative bisulfite or bisulfite conversion. Recovered mass of ssDNA was quantified using the Qubit ssDNA

quantification kit. Recoveries are shown in Figure 4, bars are labeled with the average % recovery (n=2), error bars show the standard deviation from the mean.

Figure 5: Post Bisulfite ssDNA Recovery (DNA binding time)

50kb Promega Human Genomic DNA was bisulfite converted and purified direct from the bisulfite reaction on magnetic beads using the salt-free BB2 buffer method. The input mass for all conversions was 500 ng. Binding times after addition of the BB2 solution to the bisulfite reaction mix were varied across the range 5, 10, 20 and 30 minutes. The relative concentration of magnetic beads within the BB2 solution was also tested (IX, 0.5X and 0.25X bead dilutions relative to the standard BB2 solution, representing final bead concentrations of 0.06% v/v, 0.03% v/v and 0.015% v/v respectively). The experiments were conducted in duplicate for each binding time point and each bead

concentration. The recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit. The PEG-10000 concentration of BB2 used in this series of experiments was 15% w/v.

Recoveries are shown in Figure 5, bars are labeled with the average % recovery (n=2), error bars show the standard deviation from the mean.

Figure 6: Post Bisulfite ssDNA Recovery Effect of Varying % PEG- 10000

50kb Promega Human Genomic DNA was bisulfite converted and purified direct from the bisulfite reaction on magnetic beads using the salt-free BB2 buffer method. The input mass for all conversions was 700 ng . Four different BB2 solutions were tested. These were prepared by changing the %PEG-10000 in the solution to give a final (working) concentration of 10%, 15%, 20% and 25% (w/v) . Binding times after addition of the BB2 solution to the bisulfite reaction mix were for either 5 or 15 minutes . The experiments were conducted in duplicate for each binding time point and each %PEG concentration. The recovered mass of ssDNA was quantified using the Qubit ssDNA

quantification kit. Recoveries are shown in Figure 6, bars are labeled with the average % recovery (n=2), error bars show the standard deviation from the mean.

Figure 7: Post Bisulfite ssDNA Recovery Effect of Varying % PEG- 10000

50kb Promega Human Genomic DNA was bisulfite converted and purified direct from the bisulfite reaction on magnetic beads using the salt-free BB2 buffer method. The input mass for all conversions was 500 ng . The experiments were conducted in duplicate for each sample. The recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit. Recovered converted ssDNA (100 ng ea.) were electrophoresed through a 2% TBE Agarose gel (100 V, 30 minutes) and stained with gel-red. The stained gel was visualized on a UV gel imager. Sample fragmentation range observed centered at ca. 1 kb (calibrated versus Generuler lkb DNA ladder, Thermo) . Recoveries are shown in Figure 7, bars are labeled with the average % recovery (n=2) error bars show the standard deviation from the mean.

Experimental protocol

Materials

Salt-free Post Bisulfite Bead Binding Buffer (BB2)

Component Concentration

PEG-10000 30% w/v

EDTA 0.6 mM

TrisHCl 6.0 nM

SeraMag Carboxy SpeedBeads 0.06% v/v

Salt-free Post Bisulfite Bead Binding Buffer (BB2-4000) :

Component Concentration

PEG-4000 36% w/v

EDTA 1.0 mM

TrisHCl 10.0 mM

SeraMag Carboxy SpeedBeads 0.1% v/v

Salt-free Post Bisulfite Bead Binding Buffer (BB2-10000) :

Component Concentration

PEG-10000 50% w/v

EDTA 1.0 mM

TrisHCl 10.0 mM

SeraMag Carboxy SpeedBeads 0.1% v/v Desulfonation Solution :

Bisulfite and Oxidative Bisulfite Conversion Reagents:

The TrueMethyl Oxidative Conversion kit was used for all conversion reactions, according to manufacturers standard protocol .

Other miscellaneous components :

Amicon Ultra 0.5ml 30kDa Ultracentrifuge Filtration Device

Methods :

Bisulfite and Oxidative Bisulfite Conversion:

The TrueMethyl Oxidative Conversion kit was used for all conversion reactions, according to manufacturers standard protocol.

Post Bisulfite ssDNA Purification using Amicon Ultracentrifuge Device

Method as described in the TrueMethyl vl .0 User Guide.

Post Bisulfite ssDNA Purification using Magnetic Beads:

1. Thoroughly mix 1 ml of the BB2 solution with the 200 ul of the DNA bisulfite reaction mix.

2. Incubate the bead-DNA mix for 30 min at room temperature.

3. Separate the beads from the binding solution on a magnetic rack .

4. Wash the beads one time in 70% Ethanol or 80% acetonitrile . 5. Replace the washing buffer with desulfonation solution, vortex and incubate on the magnetic rack for 5 min. 6. Wash the beads twice in 70% Ethanol or 80% acetonitrile .

7. Remove the washing solution and dry the beads .

8. Add elution buffer and resuspend the bead pellet.

9. Incubate the resuspended pellet for 15 minutes at room

temperature .

10. Separate the beads from the eluted ssDNA on a magnetic

rack .

11. Remove and retain the final purified ssDNA-containing

supernatant .

Example 1 : Post Bisulfite ssDNA Recovery (Comparison of Amicon Versus Salt-free Magnetic Bead Purification)

lkb fragmented Salmon Sperm Genomic DNA (Invitrogen) was bisulfite converted and purified direct from the bisulfite reaction using either an Amicon 30kDa ultracentrifuge filtration device or with magnetic beads using the salt-free BB2 buffer method prepared with either 36% PEG-4000 (BB2-4000) or 50% PEG- 10000 (BB2-10000) .

The input mass to the bisulfite conversion was either 1000 or 100 ng. The recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit. Recoveries are shown in Figure 1, bars are labeled with the % recovery of ssDNA.

Example 2: Post Bisulfite ssDNA Recovery (50kb DNA input)

50kb Promega Human Genomic DNA was bisulfite converted and purified direct from the bisulfite reaction on magnetic beads using the salt-free BB2 buffer method.

The input mass was titrated across the range 1000 - 250 ng and the recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit.

Recoveries are shown in Figure 2, bars are labeled with the average % recovery (n=2), error bars show the standard deviation from the mean. Example 3: Post Bisulfite ssDNA Recovery (400bp DNA input)

400 bp fragmented Lambda Genomic DNA was bisulfite and

oxidative-bisulfite converted and purified direct from the bisulfite reaction on magnetic beads using the salt-free BB2 buffer method.

The input mass was titrated across the range 800 - 50 ng and the recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit.

Recoveries are shown in Figure 3, bars are labeled with the average % recovery (n=2), error bars show the standard deviation from the mean. Bars labeled oxBS+BS report the composite recovery values from oxidative bisulfite and bisulfite converted samples .

Example 4 : Post Bisulfite ssDNA Recovery (different tissue origin)

Input DNA was either 50kb human blood leukocyte gDNA (AMSbio) or

50 kb human cerebellum gDNA (AMSbio) .

6 replicate experiments were performed with each genomic DNA type. The total input mass for each sample was 1 ^g , which was subsequently split into two separate 500 ng aliquots and each aliquot individually subjected to either oxidative bisulfite or bisulfite conversion. Recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit. Recoveries are shown in Figure 4, bars are labeled with the average % recovery (n=2), error bars show the standard deviation from the mean.

Example 5: Post Bisulfite ssDNA Recovery (DNA binding time) 50kb Promega Human Genomic DNA was bisulfite converted and purified direct from the bisulfite reaction on magnetic beads using the salt-free BB2 buffer method.

The input mass for all conversions was 500 ng. Binding times after addition of the BB2 solution to the bisulfite reaction mix were varied across the range 5, 10, 20 and 30 minutes. The relative concentration of magnetic beads within the BB2 solution was also tested (IX, 0.5X and 0.25X bead dilutions relative to the standard BB2 solution, representing final bead

concentrations of 0.06% v/v, 0.03% v/v and 0.015% v/v

respectively) . The experiments were conducted in duplicate for each binding time point and each bead concentration. The recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit.

The PEG-10000 concentration of BB2 used in this series of experiments was 15% w/v.

Example 6: Post Bisulfite ssDNA Recovery (%PEG effect)

The input mass for all conversions was 700 ng. Four different BB2 solutions were tested. These were prepared by changing the %PEG-10000 in the solution to give a final (working)

concentration of 10%, 15%, 20% and 25% (w/v) . Binding times after addition of the BB2 solution to the bisulfite reaction mix were for either 5 or 15 minutes. The experiments were conducted in duplicate for each binding time point and each %PEG concentration. The recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit.

Recoveries are shown in Figure 6, bars are labeled with the average % recovery (n=2), error bars show the standard deviation from the mean.

Example 7: Post Bisulfite ssDNA Recovery (Fragment Size

Distribution)

The input mass for all conversions was 500 ng. The experiments were conducted in duplicate for each sample. The recovered mass of ssDNA was quantified using the Qubit ssDNA quantification kit .

Recovered converted ssDNA (100 ng ea . ) were electrophoresed through a 2% TBE Agarose gel (100 V, 30 minutes) and stained with gel-red. The stained gel was visualized on a UV gel imager Sample fragmentation range observed centered at ca . 1 kb

(calibrated versus Generuler lkb DNA ladder, Thermo).

Recoveries are shown in Figure 7, bars are labeled with the average % recovery (n=2), error bars show the standard deviation from the mean.

References

1. A. M. Deaton et al Genes Dev. 25, 1010 (May 15, 2011) .

2. M. Tahiliani et al . Science 324, 930 (May 15, 2009) .

3. S. Ito et al. Nature 466, 1129 (Aug 26, 2010) .

4. A. Szwagierczak et al Nucleic Acids Res, (Aug 4, 2010) .

5. K. P. Koh et al. Cell Stem Cell 8, 200 (Feb 4, 2011) .

6. G. Ficz et al., Nature 473, 398 (May 19, 2011) .

7. K. Williams et al. Nature 473, 343 (May 19, 2011) .

8. W. A. Pastor et al. Nature 473, 394 (May 19, 2011) .

9. Y. Xu et al. MoI . Cell 42, 451 (May 20, 2011) .

10. M. R. Branco et al Nat. Rev. Genet. 13, 7 (Jan, 2012) .

11. S. Kriaucionis et al Science 324, 929 (May 15, 2009) .

12. M. Munzel et al. Angew. Chem. Int. Ed. 49, 5375 (Jul

2010) .

13. H. Wu et al. Genes Dev. 25, 679 (Apr 1, 2011) .

14. S. G. Jin et al Nuc. Acids. Res. 39, 5015 (Jul, 2011) .

15. C. X. Song et al. Nat. Biotechnol. 29, 68 (Jan, 2011) .

16. M. Frommer et al . PNAS . U.S.A. 89, 1827 (Mar 1992) .

17. Y. Huang et al . PLoS One 5, e8888 (2010) .

18. C. Nestor et al Biotechniques 48, 317 (Apr, 2010) .

19. C. X. Song et al . Nat. Methods, (Nov 20, 2011) .

20. J. Eid et al. Science 323, 133 (Jan 2, 2009) .

21. E. V. Wallace et al. Chem. Comm. 46, 8195 (Nov 21, 2010) .

22. M. Wanunu et al. J. Am. Chem. Soc, (Dec 14, 2010) .

23. WO2013/017853

24. M. J. Booth et al. Science (2012) 336, 934-937

25. M. J. Booth et al . Nature Protocols (2013) 8, 10, 1841- 1851.

26. WO2015/124955

37. Li et al Nucleic Acids (2011) Article ID 870726

38. Pfaffeneder, T. et al (2011) Angewandte. 50. 1-6

39. Lister, R. et al (2008) Cell. 133. 523-536

40. Wang et al (1980) Nucleic Acids Research. 8 (20), 4777- 4790

41. Hayatsu et al (2004) Nucleic Acids Symposium Series No. 48 (1) , 261-262 42. Lister et al (2009) Nature. 462. 315-22

43. Sanger, F. et al PNAS USA, 1977, 74, 5463

44. Bentley et al Nature, 456, 53-59 (2008)

45. KJ McKernan et al Genome Res. (2009) 19: 1527-1541

46. M Ronaghi et al Science (1998) 281 5375 363-365

47. Eid et al Science (2009) 323 5910 133-138

48. Korlach et al Methods in Enzymology 472 (2010) 431-455)

49. Rothberg et al (2011) Nature 475 348-352) .

Base Regular Bisulfite Oxidation Reduction

Sequencing Sequencing then then

Bisulfite Bisulfite

Sequencing Sequencing

C C U U U

5mC C C C C

5hmC C C U C

5fC C U U C

Table 1

Table 2

Claims

Claims :

1. A method of purifying a nucleic acid sample comprising the steps of

(ii) providing a suspension of beads in a buffer

containing polyethylene glycol and less than 1 M salt; and

(iii) mixing the population of polynucleotides with the suspension of beads, thereby binding the population of polynucleotides to the beads .

2. The method of claim 1 wherein the background salt comes from a bisulfite treatment step.

3. The method according to claim lor 2 wherein the bisulfite is ammonium, sodium or potassium bisulfite or

metabisulfite .

4. The method according to any one preceding claim wherein the beads are magnetic.

5. The method according to any one preceding claim wherein the beads have a negative charge.

6. The method according to any one preceding claim wherein the beads are carboxylate beads .

7. The method to claims 1-5 wherein the beads are silica

beads .

8. The method according to any one preceding claim wherein the polyethylene glycol is present at 5% to 50% by volume of the binding buffer. The method according to any one preceding claim wherein the suspension of beads in step (ii) contains less than 1 M sodium chloride.

The method according to any one preceding claim wherein the suspension of beads in step (ii) contains less than 0.1 M salt.

The method according to any one preceding claim wherein the suspension of beads in step (ii) contains no salt.

The method according to any one preceding claim wherein the polyethylene glycol is PEG 4000-PEG 35000.

The method according to any one preceding claim wherein the buffer has a pH of 7.0 to 9.0.

The method according to any one preceding claim wherein the buffer has a pH of 8.0.

The method according to any one preceding claim wherein the beads containing the bound polynucleotides are separated from the solution.

The method according to claim 15 wherein the beads are washed with a solution containing an organic solvent.

The method according to claim 16 wherein the beads are washed with a solution containing greater than 70% ethano or greater than 70% acetonitrile .

The method according to claims 15-17 wherein the

polynucleotides are desulfonated by exposure to a buffer with a pH greater than 10. The method according to claims 15-18 wherein the

polynucleotides are removed from the beads .

The method according to claim 19 wherein the

polynucleotides are removed using a buffer with less than 0.1 M salt or a pH >8.0.

The method according to any one of claims 1 to 20 wherein the nucleic acid sample is genomic DNA.

The method according to any preceding claim comprising the steps of:

(ii) treating a first portion of said population with bisulfite,

(iii) mixing the bisulfite treated polynucleotides with a suspension of beads in a binding buffer containing polyethylene glycol and less than 1 M salt,

(iv) separating the beads from the solution,

(v) washing the beads,

(vi) desulfonating the polynucleotides,

(vii) removing the polynucleotides from the beads,

(viii) taking a second portion of said population which has not undergone bisulfite treatment and sequencing the polynucleotides in the first and second portions of the population to produce first and second nucleotide

sequences, respectively and;

(ix) identifying the differences between the first and second nucleotide sequences, thereby identifying one or more modified cytosine residues in the sample nucleotide sequence .

A kit for use in a method of purifying a nucleic acid sample comprising;

(i) a bisulfite reagent; (ii) beads suspended in a buffer containing polyethylen glycol and less than 1 M salt.

The kit according to claim 23 wherein the binding buffe contains less than 0.1 M salt.