HYBRIDISATION COLUMN FOR NUCLEIC ACID ENRICHMENT

Abstract

The invention relates to the rapid enrichment of nucleic acid molecules of interest from complex mixtures of nucleic acids for the purpose of sequencing genes and variants, e.g. for clinical uses as well as other applications. A hybridisation column comprising an inner channel, wherein a portion of said channel is filled completely with a porous solid support comprising (a) a plurality of interconnected, micron-sized voids that permit a fluid to flow between them and the remainder of the channel, and (b) a plurality of hybridisation probes, which are bound to the surfaces of the solid support forming the voids is disclosed.

Claims

1-56. (canceled)

57. A hybridisation column comprising an inner channel, wherein a portion of said channel is filled with a porous solid support comprising: (i) a plurality of interconnected, micron-sized voids that permit a fluid to flow between them and the remainder of the channel, and (ii) a plurality of hybridisation probes, which are bound to the surfaces of the solid support forming the voids, wherein the pore structure of the solid support is homogeneous in all dimensions, wherein the solid support fills the entire cross-section of the channel.

58. The hybridisation column of claim 57, wherein the voids have an average pore size of 0.1-100 μm.

59. The hybridisation column of claim 57, wherein the portion of the channel occupied by the solid support has a volume ranging from 0.1 mm.sup.3 to 100 mm.sup.3.

60. The hybridisation column of claim 57, wherein the voids within the solid support take up about 30-50% of the volume taken up by the solid support in the channel.

61. The hybridisation column of claim 57, wherein one end of the channel comprises a porous filter, frit or permeable membrane to keep the solid support in the column when suction is applied to said end of the channel or pressure is applied to the opposite end of the channel.

62. A microfluidic device comprising one or more hybridisation column(s) according to claim 57, a temperature control element and a temperature sensor, wherein the temperature control element can be used to control the temperature within the channel of the one or more hybridisation column(s).

63. The microfluidic device of claim 62, further comprising one or more reservoir(s) wherein each reservoir is connected to one end of the channel of the one or more hybridisation column(s), wherein each reservoir can be sealed and pressurised.

64. The microfluidic device of claim 63, wherein the pressure is supplied by a source of compressed gas, which is connected to the one or more reservoir(s) via tubing.

65. The microfluidic device of claim 64, wherein pressure applied to the one or more sealed reservoir(s) pushes a fluid within the channel of the one or more hybridisation columns through the solid support.

66. The microfluidic device of claim 64, further comprising a valve within the tubing that connects the source of compressed gas supply to the one or more reservoir(s), wherein the valve can be opened and closed by a controller, which can control the flow rate of fluid driven through the channel of the one or more hybridisation column(s).

67. The microfluidic device of claim 63, wherein the pressure is supplied by a pump, wherein the pump is connected to an electronic controller, which can be programmed to control the flow rate of fluid driven through the channel of the one or more hybridisation column(s).

68. The microfluidic device of claim 63, further comprising one or more collection tubing attached to the other end of the channel of the one or more hybridisation columns.

69. The microfluidic device of claim 64, wherein one end of the channel of the one or more hybridisation columns is connected to a suction pump, wherein the suction pump is connected to a controller, which can control the flow rate of fluid driven through the channel of the one or more hybridisation column(s) when suction is applied.

70. A microfluidic device adapted for receiving the column of claim 57 in order to produce a device comprising one or more hybridisation column(s), a temperature control element and a temperature sensor, wherein the temperature control element can be used to control the temperature within the channel of the one or more hybridisation column(s).

71. A method of preparing the microfluidic device of claim 62 by inserting a hybridisation column comprising an inner channel, wherein a portion of said channel is filled with a porous solid support comprising: (i) a plurality of interconnected, micron-sized voids that permit a fluid to flow between them and the remainder of the channel, and (ii) a plurality of hybridisation probes, which are bound to the surfaces of the solid support forming the voids, wherein the pore structure of the solid support is homogeneous in all dimensions, wherein the solid support fills the entire cross-section of the channel.

72. A method for enriching nucleic acid molecules from a complex mixture of nucleic acids, wherein said method comprises: (i) providing a porous solid support comprising (a) a plurality of interconnected, micron-sized voids that permit a fluid to flow between them, and (b) a plurality of hybridisation probes, which are bound to the surfaces of the solid support forming the voids, (ii) driving the mixture of nucleic acids through the solid support thereby allowing nucleic acid molecules comprising nucleic acid sequences complementary to the nucleic acid sequences of the hybridisation probes to hybridise to the probes, wherein hybridisation is allowed to proceed for less than 10 hours, (iii) washing the solid support to remove any nucleic acids that are not hybridised to the probes, and (iv) eluting the nucleic acid molecules bound to the hybridisation probes from the solid support.

73. The method of claim 72, wherein the individual hybridisation probes for each complementary nucleic acid sequence are randomly distributed on the surface of the solid support.

74. The method of claim 72, wherein the mixture is driven through the solid support at a flow rate of 1-100 μl/minute.

75. The method of claim 72, wherein hybridisation takes place at a temperature of 55-65° C.

76. The method of claim 75, wherein the washing step is performed at a temperature 5-10° C. below the temperature used for hybridisation.

77. The method of claim 72, wherein elution is achieved by heating the solid support to a temperature of about 90-100° C.

78. The method of claim 72, wherein the hybridisation probes are 50-250-mer oligonucleotide of the same length and/or the nucleic acid molecules in the mixture have a length of 100-350.

79. The method of 72, wherein the mixture is prepared by obtaining a larger nucleic acid molecule, fragmenting it into nucleic acid fragments, and joining the fragments to adaptor molecules.

80. A method of preparing the hybridisation column of claim 57, wherein the method comprises: (i) providing a column comprising an inner channel, and (ii) filling the entire cross-section of the channel with a plurality of microbeads having about the same diameter and having linked to their surfaces a plurality of hybridisation probes, wherein the microbeads form a porous solid support.

81. The method of claim 80, wherein the diameter is 0.5 to 500 μm.

82. A kit for preparing the hybridisation columns of claim 57, wherein the kit comprises (i) one or more column(s) comprising a inner channel and (i) a container comprising a plurality of microbeads having about the same diameter and having linked to their surfaces a plurality of hybridisation probes.

Description

DESCRIPTION OF THE DRAWINGS

[0155] FIG. 1: Arrangement of microbeads in a hybridisation column for rapidly enriching DNA fragments for sequencing. (A) Microbeads (circles) are loaded into a plastic column (striped areas) with a porous frit (area filled with wavy lines) at some point along its length. The pores of the frit are smaller than the microbeads, so the fluid flow packs the microbeads into a tight bed against the frit. (B) The packed microbeads have narrow, micron-sized voids between them that permit fluid to flow (arrows) between them. (C) The small channels force DNA molecules in the flow (striped line) to come into close contact with a mixture of oligonucleotide probes on the surfaces of the packed microbeads that are complementary to the targeted DNA (solid lines) or other targets (dotted lines). A scale bar in (C) shows the approximate size of the voids when the microbeads are 5 μm in diameter. Note that the DNA fragment and probes are represented as being 5- to 10-fold longer than they are in reality (the fragments are <300 nucleotides in length and hence <100 nm).

[0156] FIG. 2: Process for enriching DNA for sequencing using microfluidic flows to accelerate hybridisation. Microbeads are functionalised with tens, hundreds, or thousands of different oligonucleotides (60-120 nt) targeted against specific regions of a genome, e.g. human, with each microbead displaying a mixture of these probes. The microbeads are loaded into the device and packed into a tight bed by fluid flow. A sample of genomic DNA is fragmented into pieces of 150-200 bp, adapters for sequencing are optionally added, and the DNA is loaded into the device upstream of the packed bed. The DNA sample is driven through the packed bed by a slow flow of hybridisation buffer at a controlled temperature (65° C.). The DNA fragments in the sample that are recognised by the oligonucleotide probes hybridise to the surface of the microbeads and are retained. The narrow channels between the microbeads accelerate this process by forcing the DNA fragments into close contact with the probes and increasing the rate of nucleation reactions. Non-targeted DNA fragments pass through the bed without hybridising to the probes, or only weakly. One or more washing steps where a wash buffer replaces the hybridisation buffer in the flow eliminate any remaining non-targeted DNA. The temperature of this step is adjusted to control the stringency of the wash(es). Finally, the targeted DNA, which is still hybridised to the probes, is eluted by raising the temperature while continuing the flow of wash buffer. This eluted DNA is then collected for sequencing. If the fragments lack adapters, then they need to be added before sequencing.

[0157] FIG. 3: Experimental data showing the selective enrichment of one oligonucleotide from a mixture of two. In this run there were 50 pmol of probe and 500 fmol of each labelled oligonucleotide.

[0158] FIG. 4: A plot of read depth versus base number through the entire genome of lambda phage. The spike peaking at base 21,986 corresponds to the region of the genome targeted by the microbead-conjugated probe.

[0159] FIG. 5: ATM gene coverage using the set-up described in Example 4. Specific, reproducible and uniform enrichment of the target regions is observed.

[0160] FIG. 6: Column design illustrating how the tapering of a column used for preparing a microbead bed can aid in achieving a microbead bed of consistent thickness and pore size and minimising any disturbance of the microbead bed during subsequent steps such as addition of the sample and washing of the microbeads. Column dimensions are given in millimetres. Panel A shows a column design with a reservoir on top which tapers into a narrow inner channel to prevent full insertion of a P100 tip, while allowing insertion of a P20 to a level 1-4 mm distant to the microbead bed to load the DNA sample. Panel B shows a column design with a tip guidance feature in a cap placed on top of the column's wide inner channel. The guidance feature contains a flange that centres both P20 and P100 tips above the location of the microbead bed at a distance sufficient to minimise any disturbance of the microbead bed during its formation and during sample loading. In addition, the column design in panel B exhibits a rim in the internal channel of the column about half way down the length of the column. The rim is placed at a sufficient distance from the top of the column so that P200 tips inserted into the guidance feature bottom out at the level where the rim is located. When wash buffer is added to the column using a P200 tip, the rim guides the tip so that the buffer runs down on the side of the wall minimising any disturbance of the microbead bed.

[0161] FIG. 7: A schematic drawing of a column suitable for practising the invention. The column (1) comprises a inner channel (2) and a porous frit (3). The inner channel (2) is stepwise tapered, dividing the column (1) into a top section (4), a neck section (5) and a bottom section (6). The frit (3) is located in the bottom section (6) and obstructs the entire cross-section of the inner channel (2). The top section (4) can be fitted with a removable cap (7), which contains an opening in the top that guides pipette tips inserted into the column (1) to the centre of the inner channel (2). The top section (4) is divided into a upper top section and a lower top section by rim (8). The inset on the left shows a rendering of the moulded column.

MODES FOR CARRYING OUT THE INVENTION

[0162] DNA molecules of interest can be enriched rapidly by passing a DNA sample through a packed bed of microbeads which are conjugated with hybridisation probes against specific sequences in the DNA molecules of interest (FIG. 1). The constrained diffusion length of the microbead bed enables the targeted DNA to be captured and enriched relative to the non-targeted DNA in a matter of minutes rather than hours.

[0163] An overview of the workflow for enriching DNA molecules of interest in a DNA sample is provided in FIG. 2. The microbeads are functionalised with tens, hundreds, or thousands of different oligonucleotides (typically 60-120mers) targeted against specific regions of a genome, e.g. the human genome, with each microbead displaying a mixture of these probes. The microbeads are loaded into a microfluidic device and packed into a tight bed by fluid flow.

[0164] A sample of genomic DNA is fragmented into pieces of 150-200 bp, adapters for sequencing are optionally added, and the DNA is loaded into the device upstream of the packed bed. The DNA sample is driven through the packed bed by a slow flow of hybridisation buffer at a controlled temperature (65° C.). The DNA fragments in the sample that are recognised by the oligonucleotide probes hybridise to the surface of the microbeads and are retained. Non-targeted DNA fragments pass through the bed, binding to the probes only weakly or not all.

[0165] One or more washing steps where a wash buffer replaces the hybridisation buffer in the flow eliminate any remaining non-targeted DNA. The temperature of this step is adjusted to control the stringency of the wash(es).

[0166] Finally, the targeted DNA, which is still hybridised to the probes, is eluted by raising the temperature while continuing the flow of wash buffer. This eluted DNA is then collected for downstream applications such as sequencing. If the fragments lack adapters, then they need to be added before sequencing.

Example 1: Rapid Enrichment of a Fluorescently-Labelled Oligonucleotide

[0167] To demonstrate and quantify the rapid enrichment of a specific sequence in a mixture of nucleic acids, two fluorescently-labelled oligonucleotides were combined and passed through a bed of microbeads functionalised with a probe complementary to one of them.

Preparation of Probe-Functionalised Microbeads

[0168] 1. A 60-mer oligonucleotide probe with a 5′ amine modification (FB-AmC6-ProbeA: 5′-TGAGGCTTGC ATAATGGCAT TCAGAATGAG TGAACAACCA CGGACCATAA AAATTTATAA-3′ (SEQ ID NO: 1); Integrated DNA Technologies, Inc.) was synthesised and diluted to 1 μM concentration in nuclease-free water. [0169] 2. 5 μm-diameter NHS-functionalised silica microbeads (Bioclone, Inc.) were resuspended in 2× Suspension Buffer (Bioclone, Inc.) to a density of 200 g/1. [0170] 3. 100 μl of the oligonucleotide solution were combined with 100 μl of the microbead suspension in a 1.5 ml microcentrifuge tube. The coupling reaction was, therefore, performed at a probe density of 5 picomoles per milligram of microbeads. [0171] 4. The tube was mounted on the rotisserie in a rotating hybridisation oven and incubated at 50° C. for 16 hours with constant rotation. [0172] 5. A magnetic rack was used to separate the microbeads from the supernatant, which was discarded. [0173] 6. The microbeads were resuspended in 200 μl 1×Washing Buffer (Bioclone, Inc.), vortexed, and placed back on the magnetic rack. Once again, the supernatant was discarded. The steps constituted one ‘wash’. [0174] 7. The microbeads were washed a further two times in 200 μl 1× Washing Buffer (Bioclone, Inc.) and twice in 500 μl nuclease-free water at 99° C. [0175] 8. The microbeads were resuspended at 20 mg/ml density in a storage buffer containing 250 mM Tris-HCl, pH 7.6, 20 mM EDTA, 0.1% Tween-20, and 0.02% sodium azide (all Sigma-Aldrich Co. LLC).

[0176] The above steps were repeated with a 10 μM solution of FB-AmC6-ProbeA in step 1 so that the microbeads were coupled with a 10-fold greater density of probe (50 pmol/mg).

Enrichment

[0177] 1. 1 mg of the probe-functionalised microbeads was loaded into a PEEK capillary with an internal bore of 381 μm (IDEX Corp.). A 0.5 μm-pore PEEK filter (IDEX Corp.) at the end of the capillary trapped the microbeads, causing them to pack into bed with a diameter of 381 μm and a length of about 10 mm [0178] 2. Hybridisation buffer was prepared as 1× Hi-RPM buffer (Agilent Technologies, Inc.)

[0179] supplemented with 50 ng/μl salmon sperm DNA (Life Technologies). The capillary was heated to 65° C. in a waterbath and the hybridisation buffer was pumped though the microbead bed at 10 μl/minute for 10 minutes using a Mitos Duo XS-Pump (Dolomite Ltd.). This incubation served to pre-hybridise (block) the microbeads and other surfaces inside the capillary. [0180] 3. A 9725i HPLC manual injection valve (IDEX Corp.) was used to introduce a 5 μl pulse of fluorescently-labelled 100-mer oligonucleotides dissolved in the same hybridisation buffer. This equimolar mixture contained 50, 500, or 5,000 fmol of each of the following two species: (i) a Cy3-labelled oligonucleotide complementary to the probe on the microbeads (FB-Cy3-TargetA: 5′-CATTAGTTCC GGCCAGCAGA TTATAAATTT TTATGGTCCG TGGTTGTTCA CTCATTCTGA ATGCCATTAT GCAAGCCTCA CAATATAGTT AAATGCAATG-3′ (SEQ ID NO: 2); Integrated DNA Technologies, Inc.) and (ii) a Cy5-labelled oligonucleotide with a non-complementary sequence (FB-Cy5-TargetB: 5′-GAGTTGCCCA TCGATATGGG CAACTCTATC TGCACTGCTC ATTAATATAC TTCTGGGTTC CTTCCAGTTG TTTTTGCATA GTGATCAGCC TCTCTCTGAG-3′ (SEQ ID NO: 3); Integrated DNA Technologies, Inc.). [0181] 4. The pulse was pumped through the microbead bed and the fluorescence of the stream flowing out of the microbead bed was measured with a 474 HPLC scanning fluorescence detector (Waters Corp.). The fluorescence signal from each of the two oligonucleotides permitted the flow-through of each species to be determined. [0182] 5. The temperature of the capillary was reduced to 25° C. and the mobile phase was switched to a wash buffer containing 0.1×SSPE (Sigma-Aldrich Co. LLC) and 0.005% (v/v)N-lauroylsarcosine (Sigma-Aldrich Co. LLC), pumped at 50 μl/minute. This washing phase helped to remove non-hybridised oligonucleotides. [0183] 6. After washing was complete, the flow rate of the wash buffer was returned to 10 μl/minute and the temperature of the capillary was raised to 65° C. to release the hybridised oligonucleotides from the surface of the microbeads. The fluorescence signal from each of the two oligonucleotide species permitted their specific capture to be quantified.

Results

[0184] A single 120-mer oligonucleotide was conjugated to an aliquot of microbeads via NHS/amine chemistry. The microbeads were packed into a short capillary terminated by a porous frit with a pore size smaller than the microbeads, trapping them inside the capillary. Hybridisation buffer was pumped through the capillary to block the microbeads.

[0185] An HPLC injection valve was used to introduce a pulse of two fluorescently-labelled oligonucleotides into a stream of hybridisation buffer. The outflow from the bed was monitored in the green and red fluorescence channels using an HPLC fluorescence detector (FIG. 3). One oligonucleotide, labelled with the green dye Cy3, was complementary to the probe oligonucleotide on the microbeads and the majority of its molecules hybridised. Some molecules were not captured and passed straight through the bed, as illustrated by the peak in outflow fluorescence between 0 and 600 seconds (striped area). The mobile phase was then switched to a cold, but stringent, washing buffer during the ‘washing’ phase. To determine how much of the labelled oligonucleotide had actually bound, the temperature was increased and the hybridised molecules melted off the probes and flowed out of the bed, as illustrated by the peak in fluorescence occurring between 1200 to 2000 seconds (stippled area). The relative areas of the two peaks indicate the fraction of targeted oligonucleotide molecules that were captured by the microbead surfaces. The other oligonucleotide, labelled with the red dye Cy5, was not complementary to the probe and did not hybridise: the vast majority of DNA flowed straight through during the ‘hybridisation’ step, leaving very little bound to the surface.

[0186] By varying the density of hybridisation probes on the microbeads and the concentration of oligonucleotides in the injected pulse it was possible to investigate the impact of each of these variables on capture efficiency (Table 1).

TABLE-US-00001 TABLE 1 Efficiency of capture of targeted and non-targeted oligonucleotides at different probe densities and oligonucleotide concentrations. Values that were not measured are indicated by ‘NM’. Moles of Moles of each injected Cy3 oligo. Cy5 oligo. Enrichment probe (pmol) oligo. (fmol) captured (%) captured (%) (fold) 5 50 31.9 NM — 5 500 11.8 NM — 5 5000 4.60 NM — 50 50 89.4 NM — 50 500 64.5 0.440 147 50 5000 9.42 NM —

[0187] The results indicate that the amount of probe on the microbeads and the amount of injected oligonucleotide both affected the overall capture of the targeted oligonucleotide. Capture efficiency was highest (89.4%) when the amount of probe was greatest (50 pmol) and the amount of injected oligonucleotide was lowest (50 fmol). Decreasing the amount of capture probe or increasing the amount of injected oligonucleotide had the effect of decreasing the capture of the targeted oligonucleotide. In the case where the capture of non-targeted oligonucleotide was also measured, the value determined was much lower than that for the targeted oligonucleotide (0.44 versus 64.5%). The ratio of these capture efficiencies indicates an overall enrichment factor of 147-fold was achieved. In all cases the hybridisation step lasted only 10 minutes.

Example 2: Rapid Enrichment of a Single Target in the Lambda Phage Genome

[0188] To demonstrate and quantify the enrichment of a specific sequence from the complete genome of an organism, a library of fragments were generated from lambda phage genomic DNA and driven through a bed of microbeads functionalised with a probe complementary to one 120 bp target.

Preparation of Probe-Functionalised Microbeads

[0189] A 120-mer oligonucleotide with a 5′ amine modification (FB-AmC6-ProbeA120: 5′-CCGTCAAAAA CATTGCATTT AACTATATTG TGAGGCTTGC ATAATGGCAT TCAGAATGAG TGAACAACCA CGGACCATAA AAATTTATAA TCTGCTGGCC GGAACTAATG AATTTATTGG-3′ (SEQ ID NO: 4); Integrated DNA Technologies, Inc.) was coupled to microbeads at 5 pmol/mg density in the same way as described in Example 1. This probe was targeted against a single region in the J02459 lambda phage reference genome (GenBank): bases 21930-22049.

Preparation of Adapter Duplexes

[0190] 1. A pair of complementary oligonucleotides were synthesised by Integrated DNA Technologies, Inc. The ‘top’ oligonucleotide was synthesised with a 5′ C3 cap and a phosphorothioate bond on the 3′ terminal base (FB-PreLig-Ad-5′-T: 5′-ACTCTTTCCC TACACGACGC TCTTCCGATC T-3′ (SEQ ID NO: 5)). The ‘bottom’ oligonucleotide was synthesised with a 5′ phosphate and 3′ C3 cap (FB-PreLig-Ad-5′-B: 5′-GATCGGAAGA GCGTCGTGTA GGGAAAGAGT-3′ (SEQ ID NO: 6)). [0191] 2. The oligonucleotides were resuspended at 30 μM concentration in a 50 μl volume of annealing buffer, containing 50 mM sodium chloride, 1 mM Tris-HCl, pH 7.5, and 100 μM EDTA (all from Sigma-Aldrich Co. LLC). [0192] 3. The mixture was denatured at 95° C. for 2 minutes, cooled slowly to 25° C. (0.1° C./second), maintained at 25° C. for 5 minutes, and then stored at 4° C. The pair of oligonucleotides annealed, creating the FB-PreLig-Ad-5′ adapter duplex. [0193] 4. The above steps were repeated with another pair of oligonucleotides to create the FB-PreLig-Ad-3′ adapter duplex. The ‘top’ oligonucleotide was synthesised with a 5′ phosphate and 3′ C3 cap (FB-PreLig-Ad-3′-T: 5′-GATCGGAAGA GCACACGTCT GAACTCCA-3′ (SEQ ID NO: 7)). The ‘bottom’ oligonucleotide was synthesised with a 5′ C3 cap and a phosphorothioate bond on the 3′ terminal base (FB-PreLig-Ad-3′-B: 5′-ACTCTTTCCC TACACGACGC TCTTCCGATC T-3′ (SEQ ID NO: 5)).

Preparation of DNA for Enrichment

[0194] 1. 2 μg of lambda phage genomic DNA (Promega Corp.) was randomly sheared into 180 bp double-stranded DNA fragments with an S2 focused ultrasonicator (Covaris Inc.), according to the manufacturer's instructions. [0195] 2. The sheared DNA was cleaned and size-selected with a 1× ratio of Agencourt AMPure XP beads (Beckman Coulter, Inc.), following the manufacturer's instructions. The elution volume was 27 μl of nuclease-free water. [0196] 3. 25 μl of the clean, sheared DNA was end-repaired and A-tailed by diluting it to 50 μl with the following components: 1×T4 DNA ligase buffer (Life Technologies), 1 mM dATP (Enzymatics, Inc.), 200 μM dNTP mixture (Enzymatics, Inc.), 100 U/ml Klenow fragment (Enzymatics, Inc.), 30 U/ml T4 DNA polymerase (Enzymatics, Inc.), 220 U/ml T4 polynucleotide kinase (Enzymatics, Inc.), and 0.3 U/μl Top DNA polymerase (Bioneer Corp.). [0197] 4. The sample was incubated at 25° C. for 30 minutes, heated to 72° C. for 30 minutes, and then cooled to 4° C. [0198] 5. 15 μl of reagents were added to the DNA sample to yield a 65 μl reaction with added ligation components: 2.3 μM FB-PreLig-Ad-5′ adapter duplex, 2.3 μM FB-PreLig-Ad-3′ adapter duplex, and 18,500 U/ml T4 DNA ligase (Life Technologies). [0199] 6. The sample was incubated at 20° C. for 15 minutes. [0200] 7. The ligated DNA was cleaned and size-selected with a 1.8× ratio of Agencourt AMPure XP beads, following the manufacturer's instructions. The elution volume was 30 μl of nuclease-free water. [0201] 8. The concentration of DNA in the clean, ligated DNA sample was determined using a Qubit dsDNA HS assay (Life Technologies). [0202] 9. The aliquoted DNA was amplified in a 50 μl ‘pre-capture’ PCR containing the following: 300 ng of clean, ligated DNA, 1× AccuBuffer (Bioline), 1 mM dNTP mixture (Bioline), 0.1/μl ACCUZYME DNA polymerase (Bioline), 1 μM forward PCR primer (FB-PCR1-5′: ACTCTTTCCC TACACGACGC TCTTCCGATC T (SEQ ID NO: 5); Integrated DNA Technologies, Inc.), and 1 μM reverse PCR primer (FB-PCR1-3′: TGGAGTTCAG ACGTGTGCTC TTCCGATCT (SEQ ID NO: 8); Integrated DNA Technologies, Inc.). [0203] 10. The sample was thermal-cycled as follows: a hot start of 98° C. for 3 minutes; 6 cycles of 98° C. for 30 seconds, 65° C. for 30 seconds, and 72° C. for 1 minute; a final extension step of 72° C. for 10 minutes; and cooled to 4° C. [0204] 11. The concentration of DNA in the clean, amplified DNA sample was determined using a Qubit dsDNA HS assay (Life Technologies).

Enrichment

[0205] 1. A hollow cylinder was milled from PTFE with a diameter of 10 mm and a length of 31 mm. An internal well with a diameter of 6 mm and a length of 10.7 mm tapered to a narrow slot with a width of about 1 mm and a length of about 10 mm. The bottom of the slot was sealed with a 0.5 μm-pore PEEK frit-in-a-ferrule (IDEX Corp.), which was screwed in from the other side of the PTFE column. A 100 μm-bore, 50 cm length of FEP capillary tubing (IDEX Corp.) connected this ferrule to a 1.5 ml microcentrifuge tube to permit collection of the outflow. [0206] 2. The column assembly was fitted into a custom rig milled from aluminium and attached to a CP-200HT-TT Peltier-based temperature controller (TE Technology, Inc.), enabling precise control of the temperature. A lid, also milled from aluminium, could be screwed onto the rig to allow the contents of the column to be pressurised with a source of compressed gas. [0207] 3. 250 μl of wash buffer containing 0.1×SSPE and 0.005% (v/v)N-lauroylsarcosine was pipetted into the well in the column. [0208] 4. The lid was closed and the headspace above the column was pressurised with nitrogen gas, pushing the buffer through the well, slot, frit, and capillary tubing into the microcentrifuge tube. A flow rate of about 5 μl/minute was maintained for 2.5 minutes to fully prime the flow path. [0209] 5. 25 μl of the previously prepared microbead suspension, equivalent to 0.5 mg, were resuspended in 10 μl of wash buffer and pipetted into the slot in the column assembly, beneath the surface of the wash buffer in the well. [0210] 6. The headspace was re-pressurised to drive the microbead suspension at a rate of about 5 μl/minute for 2.5 minutes. The frit at the base of the slot trapped the microbeads, causing them to pack into bed with a diameter of 1 mm and a depth of about 0.7 mm. [0211] 7. Wash buffer remaining in the well of the column assembly was discarded and replaced with 250 μl of hybridisation buffer consisting of 1×Hi-RPM buffer (Agilent Technologies, Inc.) and 50 ng/μl salmon sperm DNA (Life Technologies). [0212] 8. The column assembly was heated to 65° C. with the Peltier system and the headspace above was pressurised to drive the hybridisation buffer through the microbead bed at a rate of about 5 μl/minute. This flow was maintained for 10 minutes to pre-hybridise (block) the microbeads and other surfaces inside the column assembly. [0213] 9. 300 ng of the clean, amplified DNA sample were dissolved in 8 μl hybridisation buffer supplemented with 3.125% (v/v) glycerol (Sigma-Aldrich Co. LLC) and 10 μM of an oligonucleotide to block the 5′ adapter (FB-PreLig-Block-5′: 5′-ACTCTTTCCC TACACGACGC TCTTCCGATC T-3′ (SEQ ID NO: 5); Integrated DNA Technologies, Inc.) and an oligonucleotide to block the 3′ adapter (FB-PreLig-Block-3′: 5′-TGGAGTTCAG ACGTGTGCTC TTCCGATCT-3′ (SEQ ID NO: 8); Integrated DNA Technologies, Inc.). [0214] 10. The temperature of the rig was decreased to 25° C. [0215] 11. This DNA sample was denatured at 98° C. for 5 minutes, cooled to 65° C. for 5 minutes, and then stored at 4° C. for 5 minutes. The 65° C. step permitted the blocking oligonucleotides to hybridise to the adapter sequences in the DNA sample, preventing the formation of ‘daisy-chains’ that could impede enrichment. [0216] 12. 2 μl of the denatured DNA sample, containing 75 ng of DNA, were pipetted into the slot of the column assembly using a pipette. The glycerol in the sample caused it to sink through the hybridisation buffer and collect above the bed of microbeads. [0217] 13. The temperature of the rig was increased to 65° C. and the headspace was repressurised, forcing the DNA sample through the packed bed of microbeads at a flow rate of about 5 μl/minute for 10 minutes. [0218] 14. The temperature of the rig was decreased to 25° C. [0219] 15. The buffer in the well of the column assembly was replaced with 250 μl of wash buffer and the headspace was repressurised to drive the buffer through the microbeads at about 30 μl/minute for 5 minutes. [0220] 16. The previous step was repeated 4 more times with fresh wash buffer. [0221] 17. Fresh wash buffer was pipetted into the column and driven through the microbead bed at about 10 μl/minute. As the buffer flowed, the temperature of the column assembly was raised to 95° C. to release the hybridised DNA from the surface of the microbeads. During this time, the outflow from the column assembly was collected in a clean 1.5 ml microcentrifuge tube.

Recovery and Sequencing of Enriched DNA

[0222] 1. The enriched DNA was amplified in a 50 μl ‘post-capture’ PCR containing the following: 14 μl eluted DNA, 1×AccuBuffer (Bioline), 1 mM dNTP mixture (Bioline), 0.1/μl ACCUZYME DNA polymerase (Bioline), 200 nM forward PCR primer (FB-PCR2-5′: 5′-AATGATACGG CGACCACCGA GATCTACACT CTTTCCCTAC ACGACGCTCT TCCGATC-3′ (SEQ ID NO: 9); Integrated DNA Technologies, Inc.), and 200 nM reverse PCR primer (FB-PCR2-3′-Index39: 5′-CAAGCAGAAG ACGGCATACG AGATGCACTT GTGACTGGAG TTCAGACGTG TGCTC-3′ (SEQ ID NO: 10); Integrated DNA Technologies, Inc.). [0223] 2. The sample was thermal-cycled as follows: a hot start of 98° C. for 3 minutes; 16 cycles of 98° C. for 30 seconds, 65° C. for 30 seconds, and 72° C. for 1 minute; a final extension step of 72° C. for 10 minutes; and cooled to 4° C. [0224] 3. The amplified DNA was cleaned and size-selected with a 1.8×ratio of Agencourt AMPure XP beads, following the manufacturer's instructions. The elution volume was 30 μl of nuclease-free water. [0225] 4. The clean, amplified DNA was pooled with other samples and sequenced using a MiSeq Desktop Sequencer (Illumina Corp.) and a 600-cycle MiSeq Reagent Kit v3 (Illumina Corp.), following the manufacturer's instructions. [0226] 5. Reads were trimmed to 100 bases and aligned to the J02459 lambda phage reference genome using a bioinformatics pipeline based on: BWA version 0.6.2 [23], FastQC version 0.10.1 (Babraham Institute), Genome Analysis Toolkit version 1.6-13 [24], Picard version 1.104, and SAMtools version 0.1.18 [25].

Results

[0227] A single 120-mer oligonucleotide, complementary to one region of the lambda genome, was conjugated to an aliquot of microbeads via NHS/amine chemistry. These microbeads were packed into a PTFE column which integrated a porous frit with a pore size smaller than the microbeads. Hybridisation buffer was pumped through the microbead bed to block subsequent non-specific interactions.

[0228] Lambda phage genomic DNA was fragmented by sonication, end-repaired, and A-tailed. Adapter duplexes with T-overhangs were prepared by annealing complementary oligonucleotides and then ligated to the repaired fragments. These ligated fragments were then amplified by PCR and resuspended in hybridisation buffer. This sample was then pipetted onto the microbead bed and the flow was restarted, forcing the DNA between the microbeads and facilitating hybridisation of the targeted fragments. After hybridisation, non-targeted DNA was washed away by pumping wash buffer through the packed bed. DNA hybridised to the microbeads was released by raising the temperature. The outflow from the column during this elution step was collected, amplified by PCR, and then sequenced by NGS.

[0229] 486,000 paired-end reads were aligned to the lambda reference genome and used to calculate enrichment metrics. As shown in FIG. 4, plotting the number of reads against every base in the lambda genome revealed a spike corresponding to the 120 bp targeted region (GenBank J02459, 21930-22049). The average number of reads in this region +/−250 bp flanks was 32,294 reads, versus 329 for the rest of the genome. Consequently, the enrichment of the baited region was 98-fold, achieved with a 10-minute hybridisation step.

Example 3: Enrichment of Multiple Genes in the Human Genome

Preparation of Probe-Functionalised Microbeads

[0230] Microbeads were prepared in the same way as described in Example 1 with the following modification: 876 120-mer oligonucleotide probes were coupled to the microbeads at a density of 0.5 pmol/mg each. These probes were designed against 135 regions in 5 genes of the GRCh37 human reference genome (Genome Reference Consortium): ATM (chr11:108098327-108236260), BRCA1 (chr17:41197670-41276138), BRCA2 (chr13:32890573-32972932), PALB2 (chr16:23614755-23652503), and TP53 (chr17:7572902-7579937). The probes covered all exons, with 25 bp flanks, and tiled to a depth of 3×. The total targeted territory was 36.8 kb and the total baited territory was 45.5 kb.

Preparation of DNA for Enrichment

[0231] DNA was prepared in the same way as described in Example 2 with the following modification: the genomic DNA was human (Promega Corp.).

Enrichment

[0232] 1. An Oligo Clean & Concentrator column (Zymo Research Corp.) was fitted into a custom rig milled from aluminium and attached to a CP-200HT-TT Peltier-based temperature controller (TE Technology, Inc.), enabling precise control of the temperature. A lid, also milled from aluminium, could be screwed onto the rig to allow the contents of the column to be pressurised with a source of compressed gas. [0233] 2. 50 μl of the previously prepared microbead suspension, equivalent to 1 mg, was pipetted into the Zymo column. [0234] 3. The lid was closed and the headspace above the Zymo column was pressurised to 50 mbar, pushing the suspension through the column. The frit in the Zymo column caused the microbeads to collect into a bed with a diameter of about 2 mm and a depth of about 0.35 mm. The outflow of the column dripped into a 1.5 microcentrifuge tube positioned underneath. Pressure was maintained for 2.5 minutes, forcing the entire volume of buffer out of the column, but leaving the microbead bed ‘damp’. [0235] 4. A blocking buffer was prepared, containing 5.38 mM EDTA (Life Technologies), 750 ng/μl salmon sperm DNA (Life Technologies), 5.44×Denardt's solution (Life Technologies), 0.11% SDS (Life Technologies), 5.43×SSPE (Sigma-Aldrich Co. LLC), 10 μM FB-PreLig-Block-5′, and 10 μM FB-PreLig-Block-3′. 100 μl was pipetted onto the microbead bed. [0236] 5. The Zymo column was heated to 65° C. with the Peltier system and the headspace above was pressurised to 50 mbar for 2.5 minutes to drive the blocking buffer through the microbead bed at a rate of microliters/minute. This step pre-hybridised (blocked) the microbeads and other surfaces inside the Zymo column. [0237] 6. The temperature of the rig was decreased to 25° C. [0238] 7. A hybridisation buffer was prepared, containing the same components as the blocking buffer, but with 10-fold greater concentrations of the two blocking oligonucleotides (100 μM each). 750 ng of the previously prepared adapter-modified DNA sample were dissolved in 50 μl of this buffer. [0239] 8. This DNA sample was denatured at 98° C. for 5 minutes, cooled to 65° C. for 5 minutes, and then stored at 4° C. for 5 minutes. [0240] 9. 5 μl of the denatured DNA sample, containing 75 ng of DNA, were pipetted onto the microbead bed. [0241] 10. The temperature of the rig was increased to 65° C. and the headspace was repressurised to 50 mbar for 2.5 minutes, forcing the DNA sample through the packed bed of microbeads at a rate of microliters/minute. [0242] 11. The temperature of the rig was decreased to 55° C. [0243] 12. A wash buffer was prepared, containing 0.1% SDS and 0.1×SSC (Sigma-Aldrich Co. LLC). [0244] 13. 200 μl of the wash buffer, supplemented with 1 μM FB-PreLig-Block-5′, and 1 μM FB-PreLig-Block-3′, were pipetted onto the microbead bed. The headspace was repressurised to 50 mbar for 2.5 minutes to drive the buffer through the microbeads at a rate of microliters/minute. [0245] 14. The previous step was repeated 7 more times with 200 μl aliquots of wash buffer that were not supplemented with blocking oligonucleotides. [0246] 15. The temperature of the rig was decreased to 25° C. [0247] 16. Hybridised DNA was released from the microbeads by pipetting 100 μl of 100 mM NaOH (Sigma-Aldrich Co. LLC) onto the microbead bed and letting them incubate for 5 minutes. [0248] 17. The headspace was repressurised to 50 mbar for 5 minutes and the eluate was collected in a clean 1.5 ml microcentrifuge tube. [0249] 18. 100 μl of Tris-HCl, pH 7.5 (Sigma-Aldrich Co. LLC), was added to the eluted DNA to neutralise the pH. [0250] 19. The enriched DNA was cleaned using a DNA Clean & Concentrator-5 column (Zymo Research Corp.), following the manufacturer's instructions. The DNA Binding Buffer:DNA ratio used was 7 and the elution volume was 32 μl of nuclease-free water.

Recovery and Sequencing of Enriched DNA

[0251] 1. The enriched DNA was amplified in a 50 μl ‘post-capture’ PCR containing the following: 30 μl clean, enriched DNA, 1×AccuBuffer (Bioline), 1 mM dNTP mixture (Bioline), 0.1/μl ACCUZYME DNA polymerase (Bioline), 200 nM forward PCR primer (FB-PCR2-5′: 5′-AATGATACGG CGACCACCGA GATCTACACT CTTTCCCTAC ACGACGCTCT TCCGATC-3′ (SEQ ID NO: 9); Integrated DNA Technologies, Inc.), and 200 nM reverse PCR primer (FB-PCR2-3′-Index6: 5′-CAAGCAGAAG ACGGCATACG AGATGCCAAT GTGACTGGAG TTCAGACGTG TGCTC-3′ (SEQ ID NO: 10); Integrated DNA Technologies, Inc.). [0252] 2. The sample was thermal-cycled as follows: a hot start of 98° C. for 3 minutes; 16 cycles of 98° C. for 30 seconds, 65° C. for 30 seconds, and 72° C. for 1 minute; a final extension step of 72° C. for 10 minutes; and cooled to 4° C. [0253] 3. The amplified DNA was cleaned using a DNA Clean & Concentrator-5 column (Zymo Research Corp.), following the manufacturer's instructions. The DNA Binding Buffer:DNA ratio used was 5 and the elution volume was 30 μl of nuclease-free water. [0254] 4. The clean, amplified DNA was pooled with other samples and sequenced using a MiSeq Desktop Sequencer (Illumina Corp.) and a 150-cycle MiSeq Reagent Kit v3 (Illumina Corp.), following the manufacturer's instructions. [0255] 5. 75-base reads were aligned to the GRCh37 human reference genome using a bioinformatics pipeline based on: BWA version 0.6.2 [23], dsSNP build 135 [26], FastQC version 0.10.1 (Babraham Institute), Genome Analysis Toolkit version 1.6-13 [24], Picard version 1.107, and SAMtools version 0.1.18 [25], VCFtools version 0.1 [27], VEP script [28].

Results

[0256] Human genomic DNA was fragmented by sonication, end-repaired, and A-tailed. Adapter duplexes were ligated to these fragments, which were then amplified by PCR and resuspended in hybridisation buffer. This sample was then pumped through a bed of microbeads packed into a disposable commercial DNA clean-up column, which incorporates a frit. The microbeads were conjugated with 876 oligonucleotide probes against five genes in the human genome: ATM, BRCA1, BRCA2, PALB2, and TP53. After hybridisation, non-targeted DNA was washed away by pumping wash buffer through the packed bed. Targeted DNA, still hybridised to the microbeads, was released by raising the pH with sodium hydroxide. The released DNA was then collected from the column, amplified by PCR, and then sequenced by NGS.

[0257] 3,030,000 paired-end reads were aligned to the human genome and used to calculate enrichment metrics. Mean coverage in the targeted regions was 236 unique reads with 100% of the targeted bases having at least 50 reads. The percentage ‘selected bases’, i.e. base reads within the baited and near-baited (+/−250 bp) regions, was 7.16%. Consequently, the enrichment of the baited region was 4000-fold, achieved with a 2.5-minute hybridisation step.

Example 4: Enrichment of Multiple Genes in the Human Genome Using Two Hybridisation Steps

Preparation of Probe-Functionalised Microbeads

[0258] Microbeads were prepared in the same way as described in Example 3.

Preparation of DNA for Enrichment

[0259] DNA was prepared in the same way as described in Example 3.

Hybridisation 1

Preparation and Loading of the Microbeads

[0260] 1. A custom designed hybridisation column (comprising a porous, hydrophilic/polyethylene frit and a pore size 7-12 μm with a column thickness of 2.0 mm) was fitted into a custom rig and attached to a Peltier-based temperature controller enabling precise control of the temperature. A lid could be screwed onto the rig to allow the contents of the column to be pressurised with a source of compressed gas. [0261] 2. 50 μl of the previously prepared microbead suspension (20 mg/ml), was pipetted into the centre of each hybridisation column, just above the frit. [0262] 3. The lid was closed and the headspace above the column was pressurised to 50 mbar, pushing the suspension through the column. The outflow of the column dripped into a microcentrifuge tube or a 96-well plate which was positioned underneath. Pressure was maintained for 2.5 minutes, forcing the entire volume of buffer out of the column, but leaving the microbead bed ‘damp’.

Pre-Hybridisation Phase

[0263] 1. A blocking buffer was prepared, containing 5.38 mM EDTA (Life Technologies), 750 ng/μl salmon sperm DNA (Life Technologies), 5.44×Denardt's solution (Life Technologies), 0.11% SDS (Life Technologies), 5.43×SSPE (Sigma-Aldrich Co. LLC), 10 μM FB-PreLig-Block-5′, and 10 μM FB-PreLig-Block-3′. 100 μl was pipetted onto the microbead bed. [0264] 2. The column was heated to 65° C. with the Peltier system and the headspace above was pressurised to 50 mbar for 2.5 minutes to drive the blocking buffer through the microbead bed at a rate of microliters/minute. This step pre-hybridised (blocked) the microbeads and other surfaces inside the column. [0265] 3. The outflow of the column dripped into a microcentrifuge tube positioned underneath. Pressure was maintained for 2.5 minutes, forcing the entire volume of buffer out of the column, but leaving the microbead bed ‘damp’. [0266] 4. The temperature of the rig was maintained at 65° C. for the next phase of loading the DNA sample for the first hybridisation step.

Denaturing the DNA

[0267] 1. A hybridisation buffer was prepared, containing the same components as the blocking buffer, but with 10-fold greater concentrations of the two blocking oligonucleotides (100 μM each). [0268] 2. 300 ng of the previously prepared adapter-modified DNA sample was dried down and added to 5 μl of the hybridisation buffer, then briefly vortex and centrifuged. [0269] 3. This DNA sample was denatured at 98° C. for 5 minutes, cooled to 65° C. for 5 minutes, and then stored at 4° C. for 5 minutes.

Hybridisation Phase

[0270] 1. 5 μl of the denatured DNA sample, containing 300 ng of DNA, was pipetted onto the microbead bed. [0271] 2. With the temperature of the rig still at 65° C., the headspace was repressurised to 50 mbar for 2.5 minutes, forcing the DNA sample through the packed bed of microbeads.

Rinse and Wash Phase

[0272] 1. The temperature of the rig was decreased to 55° C. [0273] 2. A wash buffer was prepared, containing 0.1% SDS and 0.1×SSC (Sigma-Aldrich Co. LLC). [0274] 3. 200 μl of rinsing buffer (the wash buffer, supplemented with 1 μM FB-PreLig-Block-5′, and 1 μM FB-PreLig-Block-3′) was pipetted onto the microbead bed. The headspace was repressurised to 100 mbar for 3.5 minutes to drive the rinsing buffer through the microbeads. [0275] 4. The previous step was repeated 5 more times with 200 μl aliquots of wash buffer that were not supplemented with blocking oligonucleotides.

Elution Phase

[0276] 5. The temperature of the rig was decreased to 25° C. [0277] 6. Hybridised DNA was released from the microbeads by pipetting 100 μl of 100 mM NaOH (Sigma-Aldrich Co. LLC) onto the microbead bed and letting them incubate for 5 minutes. [0278] 7. The headspace was repressurised to 50 mbar for 5 minutes. The eluate was collected in a microcentrifuge tube or a 96-well plate block.

Neutralisation and Clean-Up of the Eluted DNA

[0279] 8. 100 μl of Tris-HCl, pH 7.5 (Sigma-Aldrich Co. LLC), was added to the eluted DNA to neutralise the pH. [0280] 9. The enriched DNA was cleaned using a DNA Clean & Concentrator-5 column (Zymo Research Corp.), following the manufacturer's instructions. The DNA Binding Buffer:DNA ratio used was 7 and the elution volume was 32 μl of nuclease-free water.

Post-Capture PCR

[0281] 1. Each of the DNA samples was amplified in a 50 μl ‘post-capture’ PCR containing the following: 30 μl clean, enriched DNA, 1×AccuBuffer (Bioline), 1 mM dNTP mixture (Bioline), 0.1/μl ACCUZYME DNA polymerase (Bioline), 200 nM forward PCR primer (FB-PCR2-5′: 5′-AATGATACGG CGACCACCGA GATCTACACT CTTTCCCTAC ACGACGCTCT TCCGATC-3′ (SEQ ID NO: 9); Integrated DNA Technologies, Inc.) and 200 nM reverse PCR primer (FB-PCR2-3′-Index1: 5′-CAAGCAGAAGA CGGCATACGA GATCGTGATG TGACTGGAGT TCAGACGTGT GCTC-3′ (SEQ ID NO: 11)). [0282] 2. The sample was thermal-cycled as follows: a hot start of 98° C. for 3 minutes; 20 cycles of 98° C. for 30 seconds, 65° C. for 30 seconds, and 72° C. for 1 minute; a final extension step of 72° C. for 10 minutes; and cooled to 4° C. [0283] 3. The amplified DNA was cleaned using a DNA Clean & Concentrator-5 column (Zymo Research Corp.), following the manufacturer's instructions. The DNA Binding Buffer:DNA ratio used was 5 and the elution volume was 30 μl of nuclease-free water. [0284] 4. The concentration of DNA in the clean, ligated DNA sample was determined using a Qubit dsDNA HS assay (Life Technologies). [0285] 5. A High-Sensitivity D1K ScreenTape was used to determine the peak fragment size which should be between 270 and 320 bp.

Hybridisation 2

[0286] The enrichment process of hybridisation 1 was repeated using 300 ng of the amplified, cleaned and enriched DNA recovered from hybridisation 1. The recovered sample was subjected to a further round of post-capture PCR.

Sequencing of Enriched DNA

[0287] The clean, amplified and enriched DNA recovered from hybridisation 2 was pooled and sequenced using a MiSeq Desktop Sequencer (Illumina Corp.) and a 150-cycle MiSeq Reagent Kit v3 (Illumina Corp.), following the manufacturer's instructions.

Results

[0288] Human genomic DNA was fragmented by sonication, end-repaired and A-tailed. Adapter duplexes were ligated to these fragments, which were then amplified by PCR and resuspended in hybridisation buffer. The samples were then pushed under pressure through a bed of microbeads compressed on top of a frit. Custom made columns were used which incorporate a Porex frit (Frit Make: Porex, Pore size 7-12 μm, Thickness—2.0 mm, Material—XS-82591). The custom made columns replaced the previously used disposable commercial DNA clean-up columns. The microbeads were conjugated with 876 oligonucleotide probes against five genes in the human genome: ATM, BRCA1, BRCA2, PALB2 and TP53. Each hybridisation was only 150 sec long. After hybridisation, non-targeted DNA was washed away by pushing wash buffer through the packed bead bed under pressure. Targeted DNA, still hybridised to the microbeads, was released by raising the pH with sodium hydroxide. The released DNA was then collected from the column, cleaned and amplified by PCR.

[0289] Two rounds of hybridisation were used. In the first round of hybridisation, the targeted DNA was hybridised to the microbeads and the non-targeted DNA was washed away. The targeted DNA was released by raising the pH, collected from the column and amplified by PCR. In the second round of hybridisation the targeted DNA was the amplified product from hybridisation 1 and the protocol followed the same steps as above.

[0290] An initial data set was generated with HapMap DNA samples (http://hapmap.ncbi.nlm.nih.gov/hapmappopulations.html). All samples met the clinical metrics for the tested 5-gene panel (48 samples/MiSeq run, target coverage of >99% bases at a depth ≧30). Repeating the hybridisation step resulted in a >14,000-fold enrichment of the baited regions, i.e. a more than 3-fold improvement over the single hybridisation step used in Example 3. Enrichment of the target regions was specific, reproducible and uniform (see FIG. 5, which shows the results for the ATM gene). Mean target coverage was >600. 100.0% of the targeted bases were covered at >30× depth. The data are summarised in Table 2.

TABLE-US-00002 TABLE 2 % Target Selected Mean Bases with Total Bases Target Fold Depth ≧ Duplication Sample # Reads (%) Coverage Enrichment 30x 50x (%) 1 1,940,644 26 616 15230 100.00 99.99 13.4 2 1,654,316 29 573 16803 100.00 99.98 14.4 3 1,799,918 30 651 17483 100.00 100.00 14.0 4 1,654,248 28 563 16201 100.00 100.00 13.0 5 1,840,250 31 738 18184 100.00 100.00 9.8 6 1,577,008 29 558 17240 100.00 100.00 15.3 7 1,916,692 25 635 14815 100.00 100.00 7.7 8 1,618,434 38 740 22620 100.00 100.00 17.6

Example 5: Hereditary/Germline Variant Detection from Whole Blood DNA

Preparation of Probe-Functionalised Microbeads

[0291] Microbeads were prepared in the same way as described in Example 3.

Preparation of DNA for Enrichment

[0292] DNA was prepared in the same way as described in Example 3 with the following modification: the genomic DNA was extracted from whole blood.

Enrichment of DNA

[0293] DNA was enriched in the same was described in Example 4.

Results

[0294] The experiment of Example 4 using HapMap DNA samples was repeated with clinical DNA samples. 48 samples were run per MiSeq lane. The data from this experiment are summarised in Table 3.

[0295] Using the set-up described in Example 4, germline/hereditary variants of >50% frequency could be detected in DNA isolated from standard whole-blood DNA samples, therefore demonstrating that microbead-based hybridisation and enrichment produce sufficient sequencing depth to reliably detect germline/hereditary variants in high-quality, genomic DNA samples.

TABLE-US-00003 TABLE 3 % Target Selected Mean Bases with Total Bases Target Fold Depth ≧ Duplication Sample # Reads (%) Coverage Enrichment 30x 50x (%) 1 1,363,484 31 382.74 17748 99.87 99.85 33.40 2 1,563,518 24 399.94 13010 99.92 99.88 17.90 3 1,246,672 28 391.10 15809 99.92 99.88 17.31 4 1,317,570 28 351.87 15187 99.92 99.87 25.61 5 1,360,830 26 394.17 14662 99.93 99.89 17.19 6 1,301,994 27 346.98 14944 99.91 99.88 25.33 7 1,418,390 24 338.49 13410 99.89 99.87 25.69 8 1,284,642 26 393.81 15234 99.98 99.92 17.06

[0296] The results were independently validated with clinical samples from 45 individuals testing a subset of 3 genes (TP53, BRCA1, BRCA2) using the Illumina TruSight Cancer panel or a Fluidigm PCR panel combined with Sanger sequencing. 44 out of 45 samples met the clinical required metric. One sample only just missed these metrics due to the low concentration of the supplied DNA. Mean target coverage was about 50% lower at >300. Fold enrichment was comparable at >12,000. The data are summarised in Table 4.

TABLE-US-00004 TABLE 4 % Target Selected Mean Bases with Total Bases Target Fold Depth ≧ Duplication Metrics Reads (%) Coverage Enrichment 30x 50x (%) Min 961,960 14 174 8,277 99.61 98.87 8.5 Mean 1,376,772 22 324 12,629 99.92 99.77 22.1 Max 1,916,802 31 457 17,748 100.00 100.00 41.6

[0297] A comparison of both data sets showed, that the set-up of Example 4—when applied to high-quality, blood-derived DNA samples—was able to detect 100% of the variants that had been identified with either the Illumina TruSight Cancer Panel or the Fluidigm PCR panel and validated with Sanger sequencing were detected: 461/461 total, which included 41 unique variants across all 45 clinical samples. For example, deletions such as 9 bp deletion in the TP53 gene (c.762_770delCATCACACT) were clearly identified in the tested clinical samples.

Example 6: Somatic Variant Detection in DNA Derived from Whole Blood Preparations

Preparation of Probe-Functionalised Microbeads

[0298] Microbeads were prepared in the same way as described in Example 3 using a panel with probes designed against 21 key genes known to contain driver mutations for a range of myeloproliferative neoplasms including polycythaemia vera (PV), essential thrombocythaemia (ET) and myelofibrosis (MF). The gene content targets ‘hot-spot’ exons where clinically relevant mutations are known and every exon for tumour suppressor, hereditary and highly implicated research-related genes. The genes included were ASXL-1, CBL, CALR, CKIT, CSF3R, EZH2, IDH1, IDH2, JAK2, MPL, NRAS, KRAS, RUNX1, SETBP1, SRSF2, TP53, TET2, DNMT3A, U2AF1, SF3B1, SH2B with a total target size of 37.6 kb.

Preparation of DNA for Enrichment

[0299] DNA was prepared from HapMap DNA samples in the same way as described in Example 5.

Enrichment of DNA

[0300] DNA was enriched in the same was described in Example 4

Results

[0301] Using the set-up described in Example 4 and standard whole-blood as source of the DNA samples, the 21-gene panel described above was able to detect somatic variants of low frequency (>1%) observed in only a small percentage of reads at any locus. The results demonstrates that this set-up provides high-stringent sensitivity to detect low-frequency in good-quality DNA samples.

[0302] Enrichment resulted in a ˜10,000-fold enrichment of the baited regions, i.e. only 30% less than the enrichment of Example 4 using a germline DNA sample. Mean target coverage was ˜1000× (see Table 5). Table 6 shows the coverage across the sites of interest observed in 12 HapMap DNA samples.

[0303] The data confirm that the panel could reliably detect somatic mutations at the following sites of interest in MPNs: JAK2 (exon 12—AAs 536-547), JAK2 (V617F), MPL (W515), KIT (D816V), and TET2 (R550).

[0304] Using the same set-up, analysis of clinical research samples further showed that the panel was able to reliably detect not only SNVs but also deletions of up to 52 bp, which are particularly informative in the CALR gene.

TABLE-US-00005 TABLE 5 Mean Dupli- Sample Selected Target Fold cation # Total Reads Bases (%) Coverage Enrichment (%) 1 4,408,302 25 1008 12078 20.5 2 4,982,940 15 826 7501 6.2 3 4,630,950 24 1062 11866 18.5 4 5,257,924 27 1372 13240 18.0 5 5,792,398 21 1149 10153 17.8 6 5,146,086 18 944 8771 11.9 7 5,199,820 13 772 6664 5.6 8 5,505,042 24 1182 11395 20.5 9 5,815,684 25 1423 12247 16.6 10 6,047,848 19 1189 9492 13.0 11 4,729,418 24 1149 11733 12.8 12 5,456,088 23 1294 11446 13.4

TABLE-US-00006 TABLE 6 Name S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 MPL_W515 546 810 762 1031 964 732 700 982 1234 1055 746 987 KIT_D816V 1142 792 1075 1319 1121 898 721 1119 1375 1098 1197 1189 TET2_R550 834 617 787 1116 879 809 649 819 1144 1026 930 939 JAK2_Exon_12 892 647 833 1013 889 761 546 882 1001 823 918 800 JAK2_V617F 1083 810 1143 1334 1092 958 752 1215 1333 1158 1251 1188

Example 7: Somatic Variant Detection in DNA Derived from FFPE Tissue

Preparation of Probe-Functionalised Microbeads

[0305] Microbeads were prepared in the same way as described in Example 3.

Preparation of DNA for Enrichment

[0306] DNA was prepared in the same way as described in Example 3 with the following modification: the genomic DNA was extracted from Formalin-Fixed Paraffin-Embedded (FFPE) cancer tissues.

Enrichment of DNA

[0307] DNA was enriched in the same way as described in Example 4

Results

[0308] Using a 5-gene panel and the set-up described in Example 4, somatic variants of low frequency (>1%) from DNA derived from FFPE tissue samples could also be detected. FFPE samples are a common source of biological material for solid cancer diagnosis and scientific research, but they can be difficult to work with because of the poor quality of extracted DNA as a result of the preparation and/or fixation process which leads to severe degradation, damage and molecular or biological modification of the DNA. As a consequence, FFPE samples often yield only low quantities of usable DNA.

[0309] Despite these challenges, the set-up described in Example 4 resulted in a 6500 fold enrichment of target DNA from FFPE breast cancer tissue (see Table 7), i.e. only 50% less than the enrichment of Example 4, which used a high-quality DNA sample, and only 35% less than the enrichment of Example 5, which used whole-blood clinical DNA samples. At 24 samples per MiSeq lane, mean target coverage was ˜300-500. Coverage could be doubled if only 12 samples are run per MiSeq lane, resulting in 600-1000 target coverage which is more suitable for somatic variant calling.

TABLE-US-00007 TABLE 7 Mean Dupli- Sample Selected Target Fold cation # Total Reads Bases (%) Coverage Enrichment (%) 1 3,048,166 11 347 6772 28.2 2 5,102,910 11 515 6362 31.4

Example 8: Identification of Suitable Frits for Microbead Bed Formation

[0310] In Examples 3, the microbead bed was prepared by applying a microbead suspension to a column that had been blocked on the opposite end by a frit which retained the microbeads within the column. The microbeads were applied under pressure which aids formation of the microbead bed. The sample as well as hybridisation and wash buffers were also applied under pressure to maintain the optimal configuration of the microbead bed and to control the flow rates through the microbead bed, making it possible to optimize hybridisation and washing conditions.

[0311] Many commercially available DNA or RNA purification columns are either designed for gravity flow or to withstand centrifugation at high-speed. Most of these columns are not suitable for use with the set-up described in Example 3 because they either do not retain the sample for a sufficient amount of time (in case of gravity flow), making it difficult to control hybridisation and washing conditions, or they require high pressure to force fluids through (e g. spin columns), resulting in unsuitably low flow rates under the low pressure conditions used in Example 3.

Frit Requirements

[0312] To identify a frit material suitable for the microfluidic applications described in Example 3, several different materials from various suppliers were tested. Each material had to meet the following requirements to be included in the tests: [0313] hydrophilic to aid wetting [0314] Low DNA binding capacity [0315] suitable for use in DNA/RNA purification. [0316] DNA/RNA-free [0317] appropriate pore sizes to be able to retain beads of >5 μm size (bead size used in Example 3). [0318] able to withstand maximum temperature of 95° C.

Tests

[0319] To prepare the test material for insertion into an empty column, a cylinder was cut out with a punch (diameter: 2.5 mm) to form a frit. The newly formed frit was inserted in an empty plastic column with tweezers. The column was then placed vertically into a rig. Two tests were performed. In the first test, 500 μl of water was pipetted into the column. The water level was marked with a felt-tip pen. After 300 seconds, the column was photographed to record the water level. The tested material was considered suitable for use as a frit if no flow occurred when no pressure was applied for a period of 300 seconds.

[0320] In the second test, a vacuum pump set at 50 mbar was connected to the base of the column. 500 μl of water was pipetted into the column and the water level was marked in the column again (if required). Another 500 μl of water was pipetted into the column and the vacuum pump was turned on and a timer was started. The timer was stopped when the water level had reached the 500 μl mark and the time was recorded. The tested material was considered suitable for use as a frit if it allowed flow rates of 5-100 μl/min at 50 mbar pressure.

Results

[0321] A range of 18 frit materials were tested. Two frit types were identified that met the suitability requirements and were found to be particularly suitable for practising the invention. The frit characteristics are summarized in Table 8:

TABLE-US-00008 TABLE 8 Reference Make Pore Size Thickness Material Frit type 1 Porex  7-12 μm 2.0 mm XS-82591 Frit type 2 ROBU 4-5.5 μm 2.5 mm VitraPOR

[0322] Frit type 1 was made of a hydrophilic polyethylene sheet with small pore sizes (7-12 μm) to prevent leakage. This material is chemically compatible with 95% ethanol and 0.1N NaOH and can withstand a maximum temperature of 121° C., which makes it suitable for autoclaving. Frits prepared from this material were relatively easy to assemble into columns. Use of frit type 1 resulted in a good flow rate (30-40 μl/min) at 50 mbar pressure and good microbead bed characteristics. Despite the slightly larger pore size, it had a good retention capacity for the beads. Frit type 1 was used for the set-up described in Example 4.

[0323] Frit type 2 was made of borosilicate glass. This material can withstand temperatures of up to 515° C. and is chemically compatible with water, acids and alkalis, salt, organic substances, chlorine and bromine. This frit type showed characteristics similar to frit type 1 in terms of flow rate, microbead bed characteristics and bead retention capacity. Frits prepared from this material were solid, maintained their shape when assembled into columns and had a flat top surface after insertion in the columns.

[0324] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

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