PURIFICATION METHOD

Abstract

A filter for isolating nucleic acid from a sample and methods of isolating and purifying nucleic acid from a sample are described. The filter has a first porous region and a second porous region. The first porous region is arranged to be contacted in use by the sample before the second porous region, and the first porous region has a nominal pore size that is greater than the second porous region.

Claims

1. A filter comprising at least a first porous region and a second porous region, the first porous region being arranged to be contacted in use by a sample before the second porous region, wherein the first porous region has a nominal pore size which is greater than the nominal pore size of the second porous region and the filter is suitable for isolating nucleic acid from the sample.

2. The filter according to claim 1, wherein the nominal pore size of the second porous region is from 0.5 μm to 1.8 μm.

3. The filter according to claim 1, wherein the nominal pore size of the second porous region is from 0.5 μm to 1.3 μm.

4. The filter according to claim 1, wherein the nominal pore size of the first porous region is from 2 μm to 3 μm.

5. The filter according to claim 1, wherein the filter device comprises a plurality of first porous regions.

6. The filter according to claim 1, wherein the filter further comprises a third porous region, the third porous region being arranged to be contacted in use by the sample after the second porous region, the third porous region having a nominal pore size which is greater than the nominal pore size of the second porous region.

7. The filter according to claim 6, wherein the nominal pore size of the third porous region is substantially equal to the nominal pore size of the first porous region.

8. The filter according to claim 1, wherein at least one of the first and second porous regions comprises a silicate.

9. The filter according to claim 8, wherein the silicate comprises a silicate glass.

10. The filter according to claim 9, wherein the silicate glass comprises, or is, borosilicate glass.

11. The filter according to claim 1, wherein the first porous region and the second porous region are formed of a fibrous material.

12. The filter according to claim 11, wherein the first porous region and the second porous region are each formed of a different fibrous material.

13. The filter according to claim 1, wherein a thickness of the filter as measured in a direction of sample flow through the filter is from 1000 μm to 2000 μm.

4. The filter according to claim 1, wherein a thickness of the filter as measured in a direction of sample flow through the filter is from 1500 μm to 2000 μm.

15. A nucleic acid extraction column comprising the filter according to claim 1 for isolating nucleic acid from a sample.

16. A method of isolating nucleic acid from a sample using a filter according to claim 1, wherein the method comprises: (i) contacting a sample comprising nucleic acid with an alcohol and a salt; (ii) contacting a filter according to claim 1 with the composition comprising nucleic acid obtained following step (i) to provide a filter having nucleic acid bonded thereto; (iii) eluting at least a portion of the nucleic acid from the filter.

17-19. (canceled)

20. The method according to claim 16 wherein the nucleic acid is eluted from the filter in step (iii) by centrifugation.

21. (canceled)

22. The method according to claim 16, wherein the salt comprises a chaotropic agent.

23. (canceled)

24. A method of isolating nucleic acid from a sample, the method comprising: (i) contacting a sample comprising nucleic acid with an alcohol and a salt; and (ii) contacting a filter with the composition comprising nucleic acid obtained following step (i) to provide a filter having nucleic acid bonded thereto; and (iii) preferably, treating the filter having the nucleic acid bonded thereto with a wash solution comprising alcohol; and (iv) eluting at least a portion of the nucleic acid from the filter, wherein elution comprises incubating the filter in an elution buffer at a temperature of from 70 to 90° C.

25-38. (canceled)

39. A kit for isolating nucleic acid from a sample, the kit comprising: (i) a filter according to claim 1; (ii) a salt, and (iii) a wash agent.

Description

DESCRIPTION OF THE FIGURES

[0228] The invention will now be illustrated with reference to the following non-limiting Examples 1-6 and FIGS. 1-8 of the accompanying drawings, which show:

[0229] FIG. 1 shows a comparison of the ratio of longer nucleic acid to shorter nucleic acid isolated using filters of the invention;

[0230] FIG. 2 (A) shows a comparison of the ratio of long to short DNA obtained using the filter of the present invention and/or the method of the present invention versus the DNeasy filter and method at an incubation temperature of room temperature, (B) shows a comparison of the average base pair length of DNA obtained using the filter of the present invention and/or the method of the present invention versus the DNeasy filter and method at an incubation temperature of room temperature, (C) shows a comparison of the ratio of long to short DNA obtained using the filter of the present invention and/or the method of the present invention versus the DNeasy filter and method at an incubation temperature of 80° C. and (D) shows a comparison of the average base pair length of DNA obtained using the filter of the present invention and/or the method of the present invention versus the DNeasy filter and method at an incubation temperature of 80° C.;

[0231] FIG. 3 shows the effect of the concentration of LiCl on the ratio of large to small DNA obtained using a method according to the invention;

[0232] FIG. 4 shows the effect of different alcohols on the ratio of large to small DNA obtained using a method according to the invention;

[0233] FIG. 5 shows the effect of the temperature of the second of two elution steps upon the ratio of long to short DNA fragments obtained using a method and filter according to the present invention;

[0234] FIG. 6 shows the effect of the temperature of the second of two elution steps upon the ratio of long to short DNA fragments obtained using a method according to the present invention on different filters. 65° C. and 80° C. were tested against each filter. (A) shows the ratio for the 4F filter, (B) shows the ratio for the 2F filter, (C) shows the ratio for the DF filter, (D) shows the ratio for the 2DF filter, (E) shows the ratio for the F2D filter and (F) shows the ratio for the DFD filter;

[0235] FIG. 7 shows the effect of two elution steps having incubation temperatures of 80° C. upon the ratio of long to short DNA obtained using different filters; and

[0236] FIG. 8 shows the ability of filters of the invention to purify longer DNA relative to known size exclusion protocols.

EXPERIMENTAL METHODS AND PERFORMANCE ANALYSIS

Example 1

[0237] Ratio of DNA Extracted

[0238] The inventor decided to study the impact of filters against the invention on the ratio of long to short DNA obtained in a DNA isolation protocol. The effect of the filters of the invention was compared to filters not according to the first aspect.

[0239] The example was otherwise carried out using a method in accordance with the third aspect of the invention. Briefly, white blood cells were isolated from a 1 ml sample of horse blood and the following procedure was followed: [0240] (a) Ice-cold hypertonic solution added to sample and the sample incubated at room temperature for 5 to 10 minutes to destroy the red blood cells; [0241] (b) Sample is centrifuged at a speed of 250×G for 3 minutes; supernatant (containing unwanted red blood cell material) is discarded without disturbing the pellet (containing white blood cells); [0242] (c) Sample (pellet containing white blood cell material) is contacted with a lysis solution comprising 0.8M LiCl, 0.5% SDS, proteinase K and 10 mM EDTA and pipette to mix to lyse the sample; [0243] (d) Sample contacted with a 2M solution of the chaotropic agent GuHCl and vortex. [0244] (e) 75% isopropanol added and vortex; [0245] (f) Transfer the sample to a nucleic acid extraction column, in this embodiment a spin column comprising a filter; [0246] (g) Spin at 4,722×g for 1 min [0247] (h) Discard the flow-through [0248] (i) Add wash solution comprising LiCl and 50% ethanol to the spin column and centrifuge at 4722 g for 1 min. Discard the flow-through. [0249] (j) Add 90% ethanol to the spin column and centrifuge at 14,462×g for 3 mins. Discard the flow-through. [0250] (k) Centrifuge the spin column at 14,462×g for 1 min. Discard the flow-through. [0251] (l) Add elution buffer (10 mM Tris HCL, 0.5 mM EDTA) to the spin column and incubate at room temperature for 1 min. Elute at 1,180 g for 2 mins to obtain fraction A; and [0252] (m) Optionally repeat (l), to obtain fraction B.

[0253] Steps (a) and (b) are optional steps used to remove red blood cells from a whole blood sample. It essentially acts to pellet the white blood cells from the sample. The white blood cells are then be lysed in step (c). As the skilled person will appreciate, step (c) is also optional if lysis of the sample is not required.

[0254] Porous regions suitable for use in the present invention are detailed in Table 1. All of the porous regions in Table 1 are available from GE Life Sciences (USA). Other suitable porous regions, for example, in the form of filter sheets, will be commercially available to the skilled person.

TABLE-US-00001 TABLE 1 Grade A B D F Nominal pore 1.6 1.0 2.7 0.7 size (based on a 98% efficiency in μm)

[0255] Each of the filters can be cut to size as necessary and is formed of borosilicate glass.

[0256] To form each filter, a variety of different porous region combinations were tested (see Table 1 for information on each porous region and Table 2 for information on the filters tested). Briefly, each of the porous regions was cut to a 0.38 cm.sup.2 diameter for insertion into the spin column. Since the porous regions were in a membrane form, to form each filter the porous regions were combined together using an O-ring formed of Purell HP570R (Lyondellbasell, Milton Keynes, UK) to secure the filters together at their peripheries.

[0257] The nomenclature used indicates the positioning of each porous region relative to contact by the sample in use. Thus, for the DF filter, the D porous region is the “top” porous region, i.e. the porous region closest to the open end of the spin column and so the porous region which will be contacted first by the sample in use, while the F porous region is underneath the D porous region. For the F2D filter, the F porous region is the “top” porous region, with the 2 D porous regions underneath.

[0258] For the present experiments, F porous regions were obtained from Porex Corporation, Aachen, Germany), while D porous regions were obtained from Whatman (GE Life Sciences (USA)). However, the skilled person will appreciate that equivalent porous regions are available from a number of other commercial suppliers.

TABLE-US-00002 TABLE 2 Content of Content of Filter of the filter of the Comparative comparative invention invention filter filter DF 1 × D and 1 × F 4F 4 × F porous porous region regions 2DF 2 × D porous 2F 2 × F porous region and 1 × regions F porous region DFD 1 × D porous F2D 1 × F porous region, 1 × F region and 2 × porous region D porous and 1 × D regions porous region

[0259] FIG. 1 shows the effect of the filters of Table 2 on the ratio of long to short DNA fragments obtained using the above protocol.

[0260] The number of DNA molecules of a particular size extracted in the elution step (l) was analysed using the FEMTO Pulse parallel capillary electrophoresis instrument (Advanced Analytical Technologies, Inc., Milton Keynes, UK). This instrument calculates nanomoles/Lt for different groups of DNA smear.

[0261] The analysis calculated the number of DNA fragments for the smear brackets: 1.3 kb to 10 kb, and 10 kb to 165.5 kb. The 10 kb to 165.5 kb bracket was then divided by the 1.3 kb to 10 kb bracket to produce the ratios shown in FIG. 1. Hence, the ratio will be understood to relate to the “10 kb cut-off”. Average values are shown with standard deviation from duplicate samples.

[0262] These ratios give an indication of the relative abundance of molecules of specific sizes. The 1.3 and 165.5 kb sizes were chosen because these are the lower and higher marker of the FemtoPulse ladder. The cut-off was chosen to be 10 kb, because molecules up to that size can severely overwhelm long read sequencing read-outs. The group of 10 kb to 165.5 kb was chosen because it covers the usual range for long-read sequencing (25 to 35 kb), as externally validated together with fragments of 50 to 165.5 kb which are considered to be very long.

[0263] As the skilled person will appreciate, a ratio of more than 1 indicates a higher number of longer-length DNA fragments compared to shorter-length DNA fragments. A ratio of less than 1 indicates a greater number of shorter-length DNA fragments compared to longer-length DNA fragments.

[0264] FIG. 1A shows the ratios obtained for the comparative filters 4F and 2F as compared to a filter of the invention DF, i.e. having a porous region with a greater nominal pore size above a porous region having a smaller nominal pore size. This Figure clearly shows that using the DF filter resulted in a greater ratio (1.91 as compared to 1.10 for 4F and 0.76 for 2F) of longer to shorter DNA fragments.

[0265] FIG. 1B shows the ratios obtained for the comparative filter FD compared to a filter of the invention, DF. As for FIG. 1A, the ratio of longer to shorter DNA fragments obtained using the DF (1.96) filter was considerably greater than the ratio obtained for the FD filter (1.10).

[0266] FIG. 10 shows the ratios obtained for the comparative filters 2F and F2D as compared to filters of the invention 2DF and DFD. Both 2DF (2.84) and DFD (3.03) gave considerably greater ratios than the comparative filters (1.17 for 2F and 0.91 for F2D), with DFD obtaining the greatest ratio.

[0267] These data demonstrate that filters of the present invention provide an improved ratio of longer to shorter nucleic acid fragments when used in nucleic acid isolation protocols. All data is derived from the first of two elutions after pre-incubation for 1 minute at 65° C. (rather than room temperature as specified in step I).

Example 2

[0268] Extracted DNA Ratio and Length Comparison to DNeasy Filter

[0269] The filter of the invention, in this embodiment the DFD filter as described in Example 1 and the method as described in Example 1 were then compared to known filters and methods of the DNeasy DNA blood and tissue extraction protocol and kit (Qiagen, Germantown, Md., US). As for Example 1, samples were analysed using a FEMTO Pulse machine.

[0270] The samples were white blood cells isolated from 1 ml of horse blood.

[0271] The inventor wished to determine if the filter of the invention provided improved isolation of longer DNA regardless of the protocol used with the filter. The results are shown in FIG. 2. These results show the ratio (FIGS. 2A and C) of long to short DNA isolated and the average base pair length of isolated DNA fragments (2B and 2D) in the eluate. For this analysis the “cut off” for the ratio was 20 KB; i.e. the 20 kb to 165.5 kb bracket was then divided by the 1.3 kb to 20 kb bracket to produce the ratios shown. The average base pair length was calculated for the isolated DNA population in the 20 to 165.5 kb bracket.

[0272] For all protocols, the elution step was performed using a pre-elution incubation step of either room temperature (FIGS. 2A and 2B) or 80° C. (FIGS. 2C and 2D). The results from the second elution are presented.

[0273] The x labels define the following conditions:

[0274] FM/FM=Method according to Example 1/DFD filter

[0275] DN/DN=DNeasy protocol and reagents from kit/Dneasy filter from kit

[0276] DN/FM=DNeasy protocol and reagents from kit/DFD filter

[0277] The results show that regardless of the incubation temperature or protocol and reagents used, the DFD filter provided an improved ratio of isolated long to short DNA fragments and an increased average base pair length compared to the DNeasy filter. This shows that the filter of the present invention provides improved results with various methods of isolation. The ratio and average base pair length were further improved when the DFD filter was used with the method of the present invention as exemplified in Example 1.

[0278] For all filters and methods, the ratio obtained and the average base pair length were improved by using a pre-elution incubation temperature of 80° C. as compared to room temperature.

Example 3

[0279] Conditions

[0280] The effect of different buffers in the present protocol was then considered. FIG. 3 shows the effect of Lithium Chloride provided on the ratio of DNA isolated. For this figure, a DNA extraction protocol was carried out in accordance with the protocol of Example 1 on a 1 ml sample of horse blood, except that a concentration of either 0M LiCl or 0.8M LiCl was used in the solution of step (c) and that incubation in step (l) was carried out at 65° C. For this protocol, a 4F filter (i.e. a comparative filter) was used. In this way the effect of the method alone could be assessed.

[0281] Ratios were determined using the same cut-offs as for FIG. 1.

[0282] FIG. 3 demonstrates that the inclusion of LiCl in a solution of a method of the invention increases the ratio of long to short DNA fragments obtained. This demonstrates that the method of the invention is able to provide an improved ratio of long to short DNA fragments independently of the filter of the present invention. All data is derived from duplicate samples (white blood cells from 1 ml of horse blood) and shows results from a first elution after pre-incubation for 1 minute at 65° C. at a cut-off of 10 kb.

[0283] FIG. 4 shows the effect of different alcohols and alcohol percentages upon the ratio of DNA isolated. As for FIG. 3, for this Figure a DNA extraction protocol was carried out in accordance with the protocol of Example 1 on a 1 ml sample of horse blood, except that the incubation step (l) was carried out at 65° C. However, 75% isopropanol, 75% ethanol, 100% isopropanol and 100% ethanol were tested in step (e) of the protocol. Ratios were determined using the same cut-offs as for FIG. 1. Where the alcohol provided was 100% isopropanol or 100% ethanol, the resulting concentration of alcohol in the composition that was submitted to filtration was 37% v/v. Where the alcohol was used was 75% isopropanol or 75% ethanol, the resulting concentration of alcohol in the nucleic acid composition that was submitted to filtration was about 28.57% by volume.

[0284] The use of 75% ethanol provided the greatest ratio (1.26), followed by 75% isopropanol (1.23), 100% ethanol (1.03) and 100% isopropanol (0.57). This also demonstrates that parameters of the method of the invention affect the ratio obtained independently of the filter of the invention. All data is derived from duplicate samples (white blood cells from 1 ml of horse blood) and shows results from a first elution after pre-incubation for 1 minute at 65° C. at a cut-off of 10 kb.

Example 4

[0285] Temperature

[0286] The effect of the incubation temperature in the elution step of the method of isolating DNA was then considered.

[0287] The protocol detailed in Example 1 was used on a sample of 3 μg of DNA of a known mixture of long and short fragments. In this way the efficiency of the improved retention of the longer fragments could be directly compared to the ratio of DNA in the input sample. The filter used was the DFD filter.

[0288] In this example, two elution steps were carried out; each elution step comprised a pre-elution incubation at the temperatures indicated in FIG. 5 for 1 minute. The number of DNA molecules in the second eluate was then analysed using the FEMTO Pulse parallel capillary electrophoresis instrument. A 20 KB cut-off was used to calculate the ratio as described in Example 2.

[0289] FIG. 5 shows the ratios obtained. While the method according to the present invention improved the ratio of long to short DNA as compared to the input at all pre-elution temperatures, the increase in long to short DNA ratio was most considerable using an 80° C. pre-elution temperature.

[0290] The effect of the pre-elution temperature of 80° C. was then tested on filters of the invention and comparative filters. The results are shown in FIG. 6. For these data, the method according to Example 1 was carried out on white blood cells from 1 ml samples of horse blood. However, two elution steps were carried out, each with a pre-elution incubation at either 65 or 80° C. for 1 minute. The DNA content of the second eluate was then analysed using the FEMTO Pulse parallel capillary electrophoresis instrument. A 20 kb cut-off was used.

[0291] A) shows the ratio for the 4F filter, (B) shows the ratio for the 2F filter, (C) shows the ratio for the DF filter, (D) shows the ratio for the 2DF filter, (E) shows the ratio for the F2D filter and (F) shows the ratio for the DFD filter. For all filters, the 80° C. incubation step greatly increased the ratio of longer DNA fragments obtained, indicating that the method of the invention using an increased temperature for incubation prior to elution gives improved results regardless of the filter used. This effect is especially pronounced for the DFD filter (F).

[0292] FIG. 7 shows the ratio of long to short DNA obtained from a second eluate using different filters following two pre-eluate incubations at 80° C. For this data, a 40 kb cut-off was used, i.e. the ratio was calculated by dividing the 40 kb to 165.5 kb bracket by the 1.3 kb to 40 kb bracket. By increasing the cut-off ratio, this assists in showing which filters are of most utility in isolating the particularly long DNA fragments. The method of Example 1 was otherwise used.

[0293] FIG. 7 shows that the filters of the invention, DFD (1.48) and 2DF (1.16), gave increased ratios compared to the comparative filters 4F (0.62) and F2D (0.67). This demonstrates that the filters of the invention give improved ratios to the comparative filters at a temperature of 80° C. as well as a lower temperature of 65° C. as indicated in FIG. 1.

Example 5

[0294] Utility of the Method of the Present Invention in Purifying Nucleic Acid

[0295] In addition to improving the proportion of long DNA fragments obtained, it is useful to be able to deplete DNA fragments under a certain cut off, for example 10 kb. This further improves the purity and/or utility of DNA samples, such as library samples or DNA extracts from known nucleic acid isolation technologies that are less suitable for the optimal extraction of longer nucleic acid for long-read sequencing technologies such as ONTs and PacBio.

[0296] The capability of the present protocol and filters to exclude short DNA fragments as compared to a known size-selection SPRI (solid phase reversible immobilisation beads) bead protocol (Agencourt AMPure XP beads, Beckman Coultier) was considered. SPRI beads are recommended by ONT for use as a quick size selection process post-extraction of DNA for depletion of up to 2 kb.

[0297] The SPRI protocol was as follows: 3 μg of DNA in 50 μl is mixed with 35 μl of beads (in 10 mM Tris-HCl, 1 mM EDTA, 1.6M NaCl, 11% PEG 8000) and the solution is reversed top to bottom at room temperature for 10 mins. It is then spun briefly for a few seconds and pelleted on a magnet. The supernatant is pipetted and the pellet is washed with 200 μl of 70% EtOH. After the EtOH is removed the EtOH wash is repeated and the pellet is allowed to dry to remove any residual EtOH. The pellet is re-suspended in 50 μl of TE buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA) and incubated for 1 min at 50° C. and 5 mins at RT. The beads are then pelleted on a magnet and the supernatant is used downstream.

[0298] The ability of various filters to purify DNA was compared to the 0.7×SPRI size selection system. 3 μg of a mixture of low and high molecular weight DNA was used as an input. Briefly 3 μg of DNA in a volume of up to 100 μl of elution buffer was mixed with a premixed solution of 150 μl solution comprising 0.8M LiCl, 0.5% SDS, proteinase K and 10 mM EDTA, 175 μl of a 2M solution of the chaotropic agent GuHCl BS and 200 μl 75% Isopropanol which was immediately loaded onto the column following the standard spin protocol. Prior to each elution (the protocol having two elutions) the elution buffer was incubated for 1 minute at 80° C. The second eluate was analysed using a 20 kb cut-off. As FIG. 8 shows, all filters performed better than the SPRI with the DFD filter providing the greatest ratio of 2.03. Elevated pre-elution incubation temperatures may contribute to the improved size selection and hence purification by facilitating detachment of the smaller molecules from the matrix, therefore they may be more readily depleted.

[0299] Accordingly, the methods of the present invention may be used to isolate and/or to purify nucleic acid. When used to purify a nucleic acid sample, it may be beneficial to use the second elution. This is because, without wishing to be bound by theory, the present inventor believes that the first elution is likely to contain the smaller nucleic acid fragments which are less suitable for downstream sequencing applications.

Example 6

[0300] Sequencing Analysis

[0301] A protocol in accordance with Example 1 was carried out using a DFD filter device in a spin column to isolate DNA (using eluate B).

[0302] An LSK109 library kit (Oxford Nanopore Technologies, Oxford, UK) was then used to prepare a sequencing library from each set of DNA. The LSK109 method results in the repair of DNA ends and dA-tailing. Sequencing adapters, supplied in the LSK109 kit, are then ligated onto the prepared ends. After this a MinION flow cell (Oxford Nanopore Technologies) was loaded with the DNA library to sequence the library.

[0303] According to protocol instructions, sequencing with the MinION flow cell can be run for six hours. However, as the flow cell has a run life of approximately 48 hours, the flow cell can be left running for this length of time. This provides more nucleic acid sequences for bioinformatics analysis.

[0304] Thus, sequencing using the MinION flow cell was run for 48 hrs and the sequencing summaries for the first 6 and 48 hrs were analyzed by Nanoplot software (https://pypi.org/project/NanoPlot/) which provided the values. Q score stands for a measure of individual base call accuracy, and is usually around 10 for ONT sequencing. ONT recommends using a cut-off of 7 for analysis:

[0305] After 6 hrs of sequencing the statistics were; average length: 27,080 bp and N50: 47,606 bp and after 48 hrs of sequencing the average length: 30,096 bp and the N50: 49,590 bp.

[0306] As the skilled person will appreciate, N50 defines the assembly quality in terms of contiguity. The N50 is defined as the sequence length of the shortest contig at 50% of the total genome length.

[0307] Oxford Nanopore Technologies generally recommends using an SPRI bead step between DNA extraction and preparation of the library to remove DNA strands below 2 kb which would otherwise overwhelm the Minlon flow cell. However, this step was not required for the present method using the 2DF or DFD filter due to the ability of the 2DF or DFD filter to clean-up and size-select the DNA during extraction.

[0308] Further sequencing analysis was carried out on white blood cell samples pelleted from 2 ml of horse blood. For these experiments, DNA was extracted in parallel using a protocol in accordance with Example 1 (using eluate B). Either a 2DF or a DFD filter device was used.

[0309] An LSK109 library kit was used to prepare a sequencing library (no post-extraction SPRI step was included) followed by sequencing either 20 μl of DNA in a final volume of 60 μl (in accordance with the manufacturer's instructions), or 47 μl of DNA in a final volume of 60 μl using a Minlon flow cell for either 6 or 48 hours.

[0310] The results are shown in Table 3 below.

TABLE-US-00003 TABLE 3 20 microLt input 47 microLt input 2DF DFD column, DFD column, DFD column, column, 6 hrs 6 hrs 6 hrs 48 hrs sequencing sequencing sequencing sequencing Mean read 16556.3 15423.7 27080.8 30096.3 length (bp) Mean read 10.5 10.3 10.2 10.1 quality Median 5475 5195 18916 23076 read length (bp) Median 10.7 10.6 10.4 10.4 read quality Number of 68894 100263 54561 156879 reads N50 (bp) 39776 42388 47606 49590 Total bases 1140627470 1546425755 1477553802 4721479216 Top 5  182110 (11.6)  234475 (11.7) 235546 (9.8)  308648 (10.8) reads (bp) with quality score  181953 (10.2)  228843 (10.1) 223082 (9.5) 246632 (9.9) 174586 (9)   216574 (11)  217688 (9.8) 235546 (9.8) 168110 (8)   212367 (9.2)  211212 (11.7) 233787 (8.7) 165681 (8.9)  208977 (11.1) 208171 (9.4) 231600 (9.1)

[0311] DNA obtained using the DFD filter had a greater N50 than the 2DF filter, of ˜42 kb vs 2DF's ˜40 kb when the 20 μl input was analysed. For the 47 μl input, the N50 goes up to ˜48 and ˜50 kb for the 6 and 48 hrs runs.

[0312] These results indicate that use of the method of the present invention to isolate and purify nucleic acid can lead to improved results downstream, for example for sequencing applications. The inventor believes this is due to the increased ratio of long to short DNA fragments in the sample. In addition, the inventor has found that because of the improved ratio, there is less interference from smaller DNA fragments and so an increased volume of DNA can be used as input for the sequencing. This, in turn, provides improved sequencing results.

REFERENCES

[0313] Cavalier, et al. 2015—Cavelier L, Ameur A, Häggqvist S, Höijer I, Cahill N, Olsson-Strömberg U, Hermanson M. (2015) Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing. BMC Cancer. 15:45. doi: 10.1186/s12885-015-1046-y.