Method for nucleic acid depletion

Field of the Invention

The invention relates to methods of depleting host nucleic acid from a biological sample.

Background to the Invention

Rapid and comprehensive infectious disease diagnostics are crucial for improved patient management and in the fight against antimicrobial resistance. Rapid diagnosis of life-threatening infectious diseases such as sepsis and pneumonia is paramount. These clinical syndromes have complex aetiologies and require pathogen recognition in challenging sample matrixes e.g. blood, sputum etc.

Currently, the "gold standard" method for clinical diagnostics is microbial culture, which is labour intensive, has long turnaround times and poor clinical sensitivity. Currently available rapid molecular methods (e.g. PCR) improve turnaround time to result and sensitivity, but are limited by range and therefore rare pathogens and resistance markers can be problematic. The most applicable technology for rapid detection of microbial pathogens is nucleic acid amplification tests (NAATs). NAATs are available for sepsis diagnostics (e.g. Septifast (RTM), Roche) but complexity of use and suboptimal performance have prevented their widespread adoption. Most of the NAATs for respiratory tract infections (RTIs) focus on the detection of respiratory viruses (e.g. Biofire Filmarray Respiratory Panel, Seegene RV15). An exception is the Curetis Unyvero (RTM) test which is designed for health care associated pneumonia. NAATs, however, are not comprehensive (e.g. the Curetis test only covers 90% of the top pathogens), seeking only a pre-set range of targets, meaning that less common pathogens will be missed.

Consequently, NAAT diagnostics are an adjunct to standard bacteriology, not a replacement, and adoption is limited. A paradigm shift in diagnostics technology is urgently required - a universal diagnostic method which can detect any pathogen (e.g. viral, bacterial, fungal) and antibiotic resistance. Agnostic/shotgun metagenomic sequencing has the potential to be the technology of choice to drive this shift. Shotgun metagenomic sequencing can detect and provide relative proportions of viruses, bacteria and fungi in a sample without any prior knowledge of the microbial community present, and is increasingly being used to investigate complex metagenomes in clinical samples.

So why is shotgun metagenomics not currently being widely applied to infection diagnosis? One reason is that next generation sequencing (NGS) has traditionally been expensive, complex to perform and difficult to analyse. The development of MinlON (RTM) nanopore sequencing technology has changed the NGS landscape with cheap portable sequencers, rapid simple library preparation (15 mins) and automated real-time analysis tools. Another major barrier is the large amount of human DNA present in clinical samples, which is often several orders of magnitude greater than the pathogen DNA present. Blood is a particularly challenging matrix for NGS-based pathogen characterization due to the vast amount of human vs. pathogen nucleic acid (particularly DNA) present (ratio is typically 10 ⁸ : 1 to 10 ⁹ : 1, based upon 10 ⁶ leukocytes/ml [with ~6.6pg DNA/cell] but as few as 1-10 colony forming units [CFU] of pathogen/ml [with ~10fg DNA/cell]). A host DNA depletion of at least about 10 ⁵ , potentially resulting in a human: pathogen DNA ratio of 10 ³ : 1, is required to facilitate NGS-based pathogen characterization, a level of depletion (giving rise to pathogen nucleic acid enrichment) not achieved by methods disclosed in the art, such as commercially available pathogen DNA enrichment methods (Looxster (RTM) Enrichment kit (Analytic Jena); NEBNext (RTM) Microbiome DNA Enrichment kit (NEB); MolYsis (RTM) Basic 5 kit (Molzym)).

It is among the objects of this disclosure to address the aforementioned problems. Summary of the Invention

Accordingly, provided is a method for depleting host nucleic acid in a biological sample, said sample having been previously obtained from an animal host, said method comprising the steps of:

(a) adding a cytolysin, or an active variant thereof, to said sample; and

(b) carrying-out a process to physically deplete nucleic acid released from host cells within said sample or otherwise render such nucleic acid unidentifiable.

Preferably, step (b) comprises adding a nuclease to said sample and/or the method further comprises the step of extracting remaining nucleic acid from the sample.

Preferably, the method further comprises the step of subjecting the extracted nucleic acid to a purification process and/or further comprises the step of amplifying the extracted nucleic acid.

Preferably, the method further comprises the step of conducting a nucleic acid amplification test on the extracted nucleic acid or, preferably, conducting a sequencing process on the extracted nucleic acid.

In preferred embodiments, the cytolysin is a phospholipase, preferably a phospholipase C (PLC), more preferably is a bacterial PLC, more preferably a Group 1 PLC, most preferably PLC from Clostridium perfringens.

In preferred embodiments the biological sample is a blood sample.

In preferred embodiments the method results in at least a 10 fold, preferably at least a 10 ² fold, preferably at least a 10 ³ fold, preferably at least a 10 ⁴ fold, most preferably at least a 10 ⁵ fold depletion of host DNA originally contained within the sample. Also provided is a kit comprising i) a cytolysin, or an active variant thereof, and ii) means to physically deplete free nucleic acid within a biological sample or otherwise render such nucleic acid unidentifiable. Preferably, said cytolysin is as defined as above and/or wherein said means comprises a nuclease.

Brief description of the Figures

Figure 1 shows amplification curves of human qPCR results after various endonuclease treatments.

Figure 2 shows amplification curves of human qPCR results after endonuclease treatment with various buffer volumes.

Figure 3 shows amplification curves of human qPCR results after HL-SAN DNase and MolDNase treatment with respective buffers.

Figure 4 shows amplification curves of human qPCR results after cytolysin treatment.

Figure 5 shows amplification curves of human qPCR results showing PLC activity in different sample conditions.

Figure 6 shows amplification curves of qPCR results after PLC and HL-SAN DNase treatment on increased volumes of bacterial spiked blood; A: Human qPCR; B: E. coli qPCR; C: S. aureus qPCR.

Figure 7 shows amplification curves of human qPCR results of PLC activity after the addition of efficient mixing during host cell lysis.

Figure 8 shows amplification curves of qPCR results after altered HL-SAN DNase inactivation; A: Human qPCR; B: E. coli qPCR; C: S. aureus qPCR. Figure 9 shows Amplification curves of qPCR results for method comparison; A: Human qPCR; B: E. coli qPCR; C: S. aureus qPCR.

Figure 10 shows C. albicans genome coverage plot after C. albicans single-plex MinlON sequencing.

Detailed Description of the Invention

General

Provided herein is a method for depleting host nucleic acid (particularly RNA and/or, most preferably, DNA) in a biological sample, said sample having been previously obtained from an animal host, said method comprising the steps of:

(a) adding a cytolysin, or an active variant thereof, to said sample; and

(b) carrying-out a process to physically deplete nucleic acid released from host cells within said sample or otherwise render such nucleic acid unidentifiable.

The animal host can be a vertebrate, e.g. a bird, a fish or, preferably, a mammal, most preferably a human. The host may, at the time of sample collection, be alive or dead.

The biological sample can be any sample that comprises animal cells (in tissue form or otherwise). Particular (e.g. clinical) samples of interest include bile, nail, nasal/bronchial lavage, bone marrow, stem cells derived from the body, bones, non-fetal products of conception, brain, breast milk, organs, pericardial fluid, buffy coat layer, platelets, cerebrospinal fluid, pleural fluid, cystic fluid, primary cell cultures, pus, saliva, skin, fetal tissue, fluid from cystic lesions, stomach contents, hair, teeth, tumour tissue, umbilical cord blood, mucus and stem cells. Particularly preferred samples include, though, joint aspirates, faeces, urine, sputum and, especially, blood (including plasma). Preferably, the sample is in liquid form. An initial sample might need to be converted to liquid form before conducting the present methodology. A liquid sample might have a volume of between ΙΟμΙ and 100ml, preferably between ΙΟμΙ and 50ml, such as between ΙΟμΙ or ΙΟΟμΙ and 20ml (e.g. 0.2ml or 1ml).

The cytolysin causes (selective) lysis of the host cells, releasing host nucleic acid such that it can be (partially or completely) depleted. Nucleic acid within a non host cell or particle (e.g. pathogen) is essentially left intact (i.e. has not been significantly removed from the sample or digested) and identifiable, such that it can be subsequently collected and analysed and, in particular, identified (by e.g. sequencing or targeted PCR). A nucleic acid is identifiable e.g. if its sequence and/or biological origin can be ascertained. Preferably, therefore, the cytolysin is added to the sample and allowed to act for a period of time such that sufficient host cell lysis can occur. Steps (a) and (b) ("cytolysin incubation" and "depletion step") can occur simultaneously, or step (b) follows step (a).

The method of depleting host nucleic acid comprises both physical depletion and (in the context of the present technology) virtual depletion (of nucleic acid released from host cells within the sample). Physical depletion can involve e.g. digesting the nucleic acid (i.e. breaking down nucleic acid polymers to e.g. base monomers) or removing nucleic acid from the sample (e.g. by any nucleic acid capture method known to the skilled person, such as deploying nucleic acid- binding magnetic beads in the sample to bind DNA and/or RNA, which can subsequently be removed or harvested from the sample).

Virtual depletion involves rendering (released) nucleic acid unidentifiable (via, in particular, targeted PCR or, most preferably, sequencing). For DNA, this means rendering the DNA non-amplifiable (e.g. by PCR) and/or (preferably) non- sequenceable. For RNA, this means rendering the RNA non-amplifiable, non- reverse-transcribable and/or (preferably) non-sequenceable. A preferred process for such rendering (particularly for DNA) involves adding a photoreactive nucleic acid-binding dye, such as propidium monoazide (PMA) or ethidium monoazide (EMA), to the sample and inducing photoreaction. Most preferably, however, the method of depletion is via digestion of nucleic acid, most preferably via enzymatic digestion. It is therefore preferred that step (b) comprises adding a nuclease to the sample. Preferably, the nuclease is added to the sample and allowed to act for a period of time such that sufficient nucleic acid digestion can occur. Preferably, therefore, a deoxyribonuclease (DNase) and/or a ribonuclease (RNase) is added to the sample (and preferably allowed to act for a period of time such that sufficient DNA/RNA digestion can occur). The nuclease can have both DNase and RNase activity (e.g. HL-SAN DNase). Depletion of host DNA is important if analysis of non host (e.g. pathogen) DNA is to be carried out. Depletion of host RNA is important if analysis of non host (e.g.

pathogen) RNA is to be carried out, and indeed can facilitate the optimisation of DNA analysis (e.g. DNA sequencing).

In such embodiments, the method preferably further comprises the subsequent step of neutralising the (or each) nuclease (i.e. decreasing or substantially eliminating the activity of the nuclease). The skilled person will recognise a range of neutralisation options, to be selected for each depletion protocol. This might include heat inactivation or, preferably, buffer exchange (i.e. the removal of a buffer in which the nuclease is active and/or replacement with or addition of a buffer in which the nuclease is substantially inactive). Preferably, the temperature of the sample (at any/all stage(s) at/before extraction of remaining nucleic acid from the sample) is maintained at 50°C or less, preferably 45°C or less, preferably 40°C or less, to optimise subsequent release of nucleic acid from the pathogen (particularly from bacterial cells).

Further steps

In preferred embodiments, the method further comprises the step of extracting remaining (preferably non host) nucleic acid from the sample (or aliquot thereof).

Part or all of the remaining nucleic acid (particularly non host nucleic acid) will be intact and identifiable. Typically, the extraction process will involve a centrifugation step to collect, in particular, non host cells/particles (e.g. pathogens) (virus particles and/or, in particular, bacterial and/or non-animal (e.g. non-mammalian) (e.g. unicellular) eukaryotic cells, such as fungi), from which the nucleic acid can be obtained.

Centrifugation conditions can be selected such that bacterial and non-animal cells, but not virus particles, are pelleted, or such that virus particles are pelleted in addition to bacterial and non-animal cells. If the former, standard virus detection tests could be performed on the supernatant. (Indeed, prior to any addition of cytolysin, one might centrifuge a clinical sample, keep the cell-containing pellet

(for the method of the current technology), and keep the supernatant for virus detection using standard procedures, with or without enrichment using the present technology.)

Nucleic acid can be obtained from the pathogen(s) using methods known in the art, and might involve the addition of a lysis buffer, a lytic enzyme(s) (degrading or abrogating cell membranes, cell walls and/or viral capsids), and/or a protease, e.g. proteinase K. Preferred lytic enzymes include lysozyme, mutanolysin, lysostaphin, chitinase and lyticase.

Optionally, the extracted nucleic acid (or aliquot thereof) is subject to a purification process, such as one known in the art. During purification of DNA, RNase is optionally used to facilitate the optimisation of subsequent DNA sequencing. However, RNase is omitted from any purification step if non host (e.g. pathogen) RNA extraction is of interest (for e.g. subsequent RNA

sequencing) (and a DNase might be used to assist with purification).

In preferred embodiments, extracted nucleic acid (or aliquot thereof) is subject to an amplification process, such as whole genome amplification, to increase the copy number of the nucleic acid, particularly where the biological sample is a blood sample. For RNA, this might involve direct amplification or conversion of RNA to cDNA, followed by amplification of cDNA. In preferred embodiments, the method further comprises the step of conducting a nucleic acid amplification test (e.g. targeted PCR amplification process, isothermal amplification, nucleic acid sequence-based amplification (NASBA)) on the extracted nucleic acid (RNA, DNA or cDNA) (or aliquot thereof) or, preferably, conducting a sequencing process on the extracted nucleic acid (or aliquot thereof), such as (e.g. short or long read) DNA or RNA sequencing, using e.g. nanopore or Illumina (RTM) sequencing.

In the preceding embodiments, nucleic acid (particularly host nucleic acid) previously rendered unidentifiable will not be amplified by any amplification process and/or (in particular) sequenced by any sequencing process.

The new method, in comparison with methods of the prior art (e.g. the MolYsis (RTM) technique, which deploys chaotropic agents to lyse host cells prior to host nucleic acid digestion), facilitates highly improved depletion of host nucleic acid (particularly DNA), while leaving non host (e.g. pathogen, particularly bacterial) nucleic acid intact (and identifiable), leading to highly improved non host (e.g. pathogen) nucleic acid enrichment, sufficient for subsequent sequencing-based (e.g. next-generation sequencing [NGS] based) (e.g. pathogen) diagnostics. A key factor in this advance has been the ability to achieve e.g. a 5 x 10 ⁴ or greater, such as 10 ⁵ or greater (e.g. 10 ⁶ or greater), fold depletion of host DNA from within biological sample from a mammalian host, and these are preferable outcome features of the present technology (as is a fold depletion of 10 or greater, 10 ² or greater, 10 ³ or greater, 5 x 10 ³ or greater, or 10 ⁴ or greater). It is particularly preferred that host nucleic acid (e.g. DNA) is undetectable (e.g. via qPCR) following deployment of the method of the invention. In more general terms, the selective depletion of host nucleic acid enables enrichment of non host nucleic acid, and hence improved identification of non host organisms. This technology is thus applicable to fields other than medical microbiology, such as biological research, veterinary medicine/diagnostic, and agriculture/food safety The cytolysin

A cytolysin (also known as a cytolytic toxin) is a protein secreted by a microorganism, plant, fungus or animal which is specifically toxic to a heterologous cell type(s), particularly promoting lysis of target cells. Preferred cytolysins are those secreted by microorganisms, particularly by bacteria, and/or those that are toxic to an animal (e.g. mammalian) cell type(s).

The cytolysin can be a cytolysin that has a detergent effect on the target cell membrane (e.g. a 26 amino acid delta toxin produced by Staphylococcus) or pores in the target cell membrane (e.g. Alpha hemolysin from S. aureus, Streptolysin O from S. pyogenes, and Perfringiolysin O produced by C.

perfringens). See e.g. : Alpha hemolysin from S. aureus - https://www.ncbi.nlm.nih.gOv/protein/BBA23710. l (SEQ ID No. 2):

1 mktrivssvt ttlllgcilm npvanaadsd iniktgttdi gsnttvktgd lvtydkengm

61 hkkvfy sfid dknhnkkilv irtkgtiagq yrvy seegan ksglawpsaf kvqlqlpdne 121 vaqisdyypr nsidtkeyms tltygfngnv tgddsgkigg liganvsigh tlkyvqpdfk

181 tilesptdkk vgwkvifnnm vnqnwgpydr dswnpvygnq lfmktrngsm kaadnfldpn

241 kassllssgf spdfatvitm drkaskqqtn idviyervrd dyqlywtstn wkgtntkdkw

301 tdrsseryki dwekeemtn

Streptolysin O from S. pyogenes - https://www.ncbi.nlm.nih.gOv/protein/BAD77794.2 (SEQ ID No. 3):

1 msnkktfkky srvaglltaa liignlvtan aesnkqntas tettttseqp kpesseltie

61 kagqkmddml nsndmiklap kemplesaek eekksedkkk seedhteein dkiyslnyne

121 levlaknget ienfvpkegv kkadkfivie rkkkninttp vdisiidsvt drtypaalql

181 ankgftenkp davvtkrnpq kihidlpgmg dkatvevndp tyanvstaid nlvnqwhdny

241 sggntlpart qytesmvysk sqieaalnvn skildgtlgi dfksiskgek kvmiaaykqi

301 fytvsanlpn npadvfdksv tfkdlqrkgv sneapplfvs nvaygrtvfv kletssksnd 361 veaafsaalk gtdvktngky sdilenssft avvlggdaae hnkvvtkdfd virnvikdna

421 tfsrknpayp isytsvflkn nkiagvnnrt eyvettstey tsgkinlshq gayvaqyeil

481 wdeinyddkg kevitkrrwd nnwysktspf stviplgans rnirimarec tglawewwrk

541 viderdvkls keinvnisgs tlspygsity k Preferably, the cytolysin is a cytolysin that digests a cell membrane component,

(e.g. phospholipids, i.e. is a phospholipase). An example is Sphingomylinease (also know as beta-toxin) from S. aureus, see e.g.

https://www.ncbi.nlm.nih.gOv/protein/CAA43885. l (SEQ ID No. 4): 1 mmvkktksns lkkvatlala nlllvgaltd nsakaeskkd dtdlklvshn vymlstvlyp

61 nwgqykradl igqssyiknn dvvifneafd ngasdkllsn vkkeypyqtp vlgrsqsgwd

121 ktegsysstv aedggvaivs kypikekiqh vfksgcgfdn dsnkgfvytk iekngknvhv

181 igthtqseds rcgaghdrki raeqmkeisd fvkkknipkd etvyiggdln vnkgtpefkd

241 mlknlnvndv lyaghnstwd pqsnsiakyn ypngkpehld yiftdkdhkq pkqlvnevvt 301 ekpkpwdvya fpyyyvyndf sdhypikays k

The phospholipase can be a phospholipase A, B, C or D, such as PLD from Streptomyces, see e.g. https://www.ncbi.nlm.nih.gOv/protein/BAL15170. l (Streptomyces vinaceus) (SEQ ID No. 5):

1 mhrhtpslrr psahlpsala vraavpaall alfaavpasa apaagsgadp aphldaveqt 61 lrqvspgleg qvwertagnv ldastpggad wllqtpgcwg ddkctarpgt eqllskmtqn 121 isqatrtvdi stlapfpnga fqdaivsglk tsaargnklk vrvlvgaapv yhlnvlpsky 181 rdelvaklga darnvdlnva smttsktafs wnhskllvvd gqsvitggin dwkddyleta 241 hpvadvdlal rgpaaasagr yldelwswtc qnksniasvw fassngaacm pamakdtapa 301 apapapgdvp avavgglgvg ikrndpsssf rpalpsapdt kcvvglhdnt nadrdydtvn 361 peesalrtli ssanrhieis qqdvnatcpp lprydirvyd alaarmaagv kvrivvsdpa 421 nrgavgsggy sqikslseis dtlrdrlalv tgdqgaakat mcsnlqlatf rssqsptwad 481 ghpyaqhhkv vsvddsafyi gsknlypawl qdfgyvvesp aaaaqlnarl lapqwqysra 541 tatidheral cqs

Preferably the phospholipase is a phospholipase C (PLC) (i.e. a phospholipase that cleaves before the phosphate, releasing diacylglycerol and a phosphate- containing head group). Preferably the PLC is a bacterial PLC, selected from any of the following groups:

Group 1 - Zinc metallophospholipases

Group 2 - Sphingomyelinases (e.g. sphingomyelinase C)

Group 3 - Phosphatidylinositol

Group 4 - Pseudomonad PLC

A Group 1 PLC is preferred, particularly PLC from Clostridium perfringens, see e.g. https://www.ncbi.nlm.nih.gOv/protein/EDT77687. l (SEQ ID No. l):

1 mkrkickali caalatslwa gastkvyawd gkidgtgtha mivtqgvsil endmsknepe 61 svrknleilk enmhelqlgs typdydknay dlyqdhfwdp dtdnnfskdn swylaysipd 121 tgesqirkfs alary ewqrg nykqatfylg eamhyfgdid tpyhpanvta vdsaghvkfe 181 tfaeerkeqy kintagcktn edfyadilkn kdfnawskey argfaktgks iyyshasmsh 241 swddwdyaak vtlansqkgt agyiyrflhd vsegndpsvg knvkelvayi stsgekdagt 301 ddymyfgikt kdgktqewem dnpgndfmtg skdtytfklk denlkiddiq nmwirkrkyt 361 afpdaykpen ikviangkvv vdkdinewis gnstynik

This cytolysin provides for highly effective lysis of animal host cells in the present technology, despite reports in the literature that purified C. perfringens PLC when used alone has no cytotoxic activity against leukocytes.

The cytolysin can be a wild-type cytolysin or an active variant (produced e.g. by recombinant DNA technology). An active variant of a cytolysin is a variant of a cytolysin that retains the ability to lyse a target cell, demonstrating e.g. at least 10%, preferably at least 25%, preferably at least 50%, preferably at least 60%>, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% of the activity of the wild-type protein in any assay where lytic activity against a target cell can be shown for the wild-type protein.

"An active variant thereof includes within its scope a fragment of the wild-type protein. In preferred embodiments, a fragment of the wild-type protein is selected that is at least 10% of the length of the wild-type protein sequence, preferably at least 20%), preferably at least 30%>, preferably at least 40%, preferably at least 50%), preferably at least 60%>, preferably at least 70%, preferably at least 80%>, preferably at least 90% and most preferably at least 95% of the length of the wild- type protein sequence.

"An active variant thereof also includes within its scope a protein sequence that has homology with the wild-type protein sequence, such as at least 50% identity, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, and most preferably at least 99% identity, for example over the full wild-type sequence or over a region of contiguous amino acid residues representing 10% of the length of the wild-type protein sequence, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% and most preferably at least 95% of the length of the wild-type protein sequence. Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as "hard homology").

The homologous active cytolysin variant typically differs from the wild-type protein sequence by substitution, insertion or deletion, for example from 1, 2, 3, 4, 5 to 8 or more substitutions, deletions or insertions. The substitutions are preferably 'conservative', that is to say that an amino acid may be substituted with a similar amino acid, whereby similar amino acids share one of the following groups: aromatic residues (F/H/W/Y), non-polar aliphatic residues (G/A/P/I/L/V), polar-uncharged aliphatics (C/S/T/M/N/Q) and polar-charged aliphatics (D/E/K/R). Preferred sub-groups comprise: G/A/P; I/L/V; C/S/T/M; N/Q; D/E; and K/R.

The cytolysin or active variant (as described above) may have any number of amino acid residues added to the N-terminus and/or the C-terminus provided that the protein retains lytic activity. Preferably, no more than 300 amino acid residues are added to either or both ends, more preferably no more than 200 amino acid residues, preferably no more than 150 amino acid residues, preferably no more than 100 amino acid residues, preferably no more than 80, 60 or 40 amino acid residues, most preferably no more than 20 or 10 or 5 amino acid residues.

Preferably, the sample is subject to mixing after the cytolysin has been added.

Preferably, to promote cytolysin activity, particular buffering conditions and/or incubation temperature might be provided for any one selected cytolysin.

Cytolysin incubation can take place at e.g. between 5°C and 50°C, such as between 15°C and 45°C (e.g. 37°C), and for between lmin and 120min, preferably between lmin and 60min, more preferably between lmin and 30min (e.g. 15min or 20min). For part or all of the cytolysin incubation, the sample is preferably subject to mixing/shaking, at e.g. between 1 and 1500rpm, preferably between 1 and lOOOrpm (e.g. at 500rpm or lOOOrpm).

Preferably, the cytolysin is used in the sample at a concentration of at least O. lmg/ml, such as between O. lmg/ml and lOOmg/ml, preferably between

O. lmg/ml and lOOmg/ml, preferably between lmg/ml and lOOmg/ml (e.g. at 40mg/ml).

The DNase

If a DNase is used in the present methodology, the DNase can be an endonuclease or an exonuclease (or a combination thereof can be provided), preferably an endonuclease.

Preferred DNases (particularly where the biological sample is a blood sample) include HL-SAN DNase (heat labile salt activated nuclease, supplied by

Arcticzymes) and MolDNase (endonuclease active in the presence of chaotropic agents and/or surfactants, supplied by Molzym), and active variants are also contemplated, essentially as discussed above in relation to the cytolysin.

Preferably, the sample is subject to mixing after the DNase has been added.

Preferably, to promote DNase activity, particular buffering conditions and/or incubation temperature might be provided for any one selected DNase. DNase incubation can take place at e.g. between 5°C and 50°C, such as between 15°C and 45°C (e.g. 37°C), and for between lmin and 120min, preferably between lmin and 60min, more preferably between lmin and 30min (e.g. 15min). In particularly preferred embodiments, the DNase buffer is added to the sample, containing the cytolysin, and incubated (e.g. as described above) before pelleting. The pellet is then resuspended in DNase buffer and the DNase itself is added (ahead of further incubation). The biological sample

Preferably, the biological sample is a blood sample. Preferably, where the sample is blood, the cytolysin targets/lyses (e.g. human) leukocytes. Preferably, especially where the sample is blood and/or the cytolysin is PLC from

Clostridium perfringens, the sample comprises a chelating agent (e.g. EDTA).

Kits Also provided is a kit comprising a cytolysin (according to e.g. any of the aspects described above) (preferably with a buffer for the cytolysin) and means to physically deplete free nucleic acid within a biological sample or otherwise render such nucleic acid unidentifiable. Free nucleic acid includes nucleic acid not contained within a cell or virus particle (e.g. has been released/liberated from animal cells within the sample as a result of lysis of those cells).

The means can be e.g means for nucleic acid capture (using e.g. magnetic bead technology), means for rendering nucleic acid unidentifiable (e.g. PMA or EMA) or, preferably, a nuclease (e.g. a DNase) (preferably with a suitable buffer and/or a composition for inactivating the nuclease), according e.g. to any of the aspects described above.

General

Please note that wherever the term 'comprising' is used herein we also

contemplate options wherein the terms 'consisting of or 'consisting essentially of are used instead. In addition, please note that the term 'protein' used herein can be used interchangeably with the term 'polypeptide'.

Examples

In the context of medical microbiology, metagenomics sequencing needs to achieve sufficient genome coverage to identify the pathogenic species present and preferably detect all resistance markers, whether mutational or acquired. To deliver this we estimate that a minimum of lOx genome coverage is required. We directly sequenced (HiSeq) blood, spiked with pathogen cells {Escherichia coli), which delivered human reads only, highlighting the need for pathogen DNA enrichment (data not shown). Hence, host DNA depletion is required to reliably and cost effectively apply metagenomics to infectious disease diagnosis.

Here, we describe the process of developing a simple, rapid and highly efficient human DNA depletion method to enable downstream metagenomic sequencing (and other molecular applications e.g. PCR) for the detection and identification of pathogens and associated antibiotic resistance markers.

For efficient and cost effective metagenomic diagnosis of infection, human DNA depletion or pathogen DNA enrichment is essential. We took the human DNA depletion approach focussing on differential lysis of human cells, and removal of human DNA, leaving intact non-human pathogens for further analysis. We used blood as a model sample type, as blood represents one of the most complex clinical samples to successfully apply metagenomic infection diagnosis due to the very high ratio of human: pathogen DNA (as high as 10 ⁹ : 1). We applied cytolysins for differential lysis of human cells and endonucleases

(DNases) for digestion of liberated DNA. We tested a number of DNases to determine the most efficient in blood. We then combined the most efficient DNases with various cytolysins to determine whether and how efficiently these toxins would lyse the DNA-containing leukocytes in blood.

A positive control (PC) was added in to every experiment, which was DNA extracted from 200μ1 of blood. For cytolysin experiments, the blood was spiked with appropriate pathogen entities, e.g. the most common sepsis causing pathogens (E. coli and S. aureus), C. albicans, A. niger, HB V, or HIV, to ensure that pathogens were not lysed during the procedure. For all qPCR reactions, a no template control (NTC; molecular grade nuclease free dH ₂ 0) was included. A MolDNase control sample (from the MolYsis (RTM) kit, Molzym, Germany) was also included where appropriate as it has been proven to work in blood.

Subsequently, DNA was extracted as follows (unless otherwise stated in the experimental procedure):

1. Bacterial lysis buffer (to a maximum volume of 380μ1) and proteinase K (20μ1) was added to the treated sample and mixed by vortexing. No bacterial lysis buffer was added to blood samples that were not spiked with bacteria (volume made up to 400 μΐ with PBS where necessary).

2. All samples were incubated at 65°C for 5min

3. Followed by purification on the MagNAPure (RTM)

For all experiments, human and non human nucleic acid was quantified using qPCR. Specific hydrolysis probe assays were designed or taken from the literature to detect human, E. coli and S. aureus DNA (all were single copy gene targets;

RNA polymerase II, cyaA and eap respectively). In addition, fungal and viral targets included C. albicans 5.8S rRNA, A. niger ITS 1-2, HBV X gene, and HIV 5' nuclease assay in LTR gene. All qPCR results are presented as amplification curves and/or quantification cycle (Cq) values (this represents the cycle at which the fluorescence signal increases above background which is directly related to the quantity of starting template concentration). The relative concentration of DNA in samples was calculated using the ACq (every 3.3 cycles represents a 10-fold difference in concentration; the higher the Cq value the less starting template DNA was present in the sample). Example 1 - Efficacy of endonucleases for DNA digestion in blood

Initial focus was on identifying an endonuclease that would digest DNA released from leukocytes so that the efficacy of cytolysins could be easily assessed in blood. In this experiment, blood samples were freeze thawed three times to release human DNA and an endonuclease; either DS-DNase, HL-SAN DNase (heat labile, salt active nuclease) or micrococcal nuclease from S.aureus was added, incubated at 37°C and DNA was extracted. Controls included a positive control (PC - DNA from 200μ1 spiked blood without DNase treatment), a MolDNase control (known to work in blood) and a negative control (NTC - nuclease free water), as detailed above. Human specific qPCR was performed on all DNA extracts and Cq values were compared to determine whether the endonuclease treatment worked.

Detailed procedure:

1. To lyse blood cells, samples were frozen at -70°C and thawed at room temperature (RT) three times

2. Freeze-thawed blood was aliquoted into 5x 200μ1 samples

3. To sample 1, 5μ1 of HL-SAN DNase (28.4υ/μ1) was added

4. To sample 2, 5μ1 of DS-DNase (2υ/μ1) was added

5. To sample 3, 20μ1 of nuclease micrococcal (resuspended in ΙΟΟμΙ of

nuclease free water; 0.62υ/μ1) was added

6. All samples were mixed by vortexing

7. Samples 1-3 and PC were incubated at 37°C for 30min

8. To the MolDNase control sample, 50μ1 of DB1 buffer was added followed by 5μ1 of MolDNase then incubated at RT for 15min

9. All reactions were stopped by adding 5μ1 of DNase inactivation buffer (Ambion (RTM), life technologies (RTM))

10. DNA was extracted and quantified by human qPCR (as described above)

Results:

As shown in Table 1 and Figure 1, DS-DNase (sample 2) and nuclease micrococcal (sample 3) showed no endonuclease activity on human DNA in blood samples, with ACq <1 compared with PC. With a ACq of 2.2, HL-SAN DNase (sample 1) showed endonuclease activity resulting in an approximate 4-fold reduction in human DNA when compared to the PC. As previously stated MolDNase was known to work in blood samples and showed the greatest endonuclease activity with the highest Cq value.

Table 1: Human qPCR results after various endonuclease treatments

Conclusion:

From all the endonucleases tested in this experiment, HL-SAN DNase was the only one to show the potential to work effectively in blood. HL-SAN DNase was the endonuclease of choice selected for further testing. As HL-SAN DNase is known to be most active in high salt concentrations, we aimed to test a high salt buffer to improve activity, and Example 2 details buffer optimization.

Example 2 - Optimization of HL-SAN buffer conditions

From Example 1, HL-SAN DNase was chosen as the most promising

endonuclease to work in blood. As HL-SAN DNase is a salt active enzyme, we tested the addition of a high salt buffer to optimize HL-SAN DNase activity on human DNA in blood samples. A high-salt buffer was made and added in various volumes to freeze-thawed blood samples with HL-SAN DNase, incubated at the known working temperature (37°C), DNase inhibitor was added and samples further incubated. MolDNase control, PC and NTC were included; all samples were subjected to DNA extraction and human qPCR (as detailed above).

HL-SAN buffer components:

lOmM Tris HC1, lOOmM magnesium and 1M NaCl pH8.5

Detailed procedure:

1. To lyse blood cells, 2ml of blood was frozen at -70°C and thawed at RT three times

2. Freeze-thawed blood was spiked with human DNA and aliquoted into 5x 200μ1 samples

3. To sample 1, 20μ1 of HL-SAN buffer and 3μ1 of HL-SAN DNase was added

4. To sample 2, ΙΟΟμΙ of HL-SAN buffer and 3μ1 of HL-SAN DNase was added

5. To sample 3, 180μ1 of HL-SAN buffer and 3μ1 of HL-SAN DNase was added

6. The above reactions were incubated at 37°C for 15min

7. To the MolDNase control, 50μ1 of DB1 buffer and 5μ1 MolDNase was added and incubated at RT for 15min

8. All reactions were stopped by adding 5μ1 of DNase inactivation buffer (Ambion (RTM), life technologies (RTM))

9. DNA was extracted and quantified by human qPCR (as described above)

Results:

Table 2 and Figure 2 show that the addition of HL-SAN buffer increases the activity of HL-SAN DNase in correlation with an increase in volume. The most effective amount of HL-SAN buffer was 180μ1, which resulted in a similar activity to MolDNase (<1 Cq difference between DNase treatments) and reduced the level of human DNA approximately 32-fold (ACq5) compared to no endonuclease treatment (PC). In the absence of buffer, HL-SAN DNase alone, resulted in a human qPCR Cq value of 24.77 (Table 1), with the addition of 180μ1 HL-SAN buffer this increased to 27.02Cq (Table 2), showing an increase in HL- SAN DNase activity to reduce human DNA approximately 4-fold (ACq 2).

Table 2: Human qPCR results after endonuclease treatment with various buffer volumes

Conclusion:

The addition of a high salt buffer (HL-SAN buffer) increased the efficiency of HL-SAN DNase to digest human DNA present in the blood samples after cell lysis by freeze-thawing. Using this combination (HL-SAN buffer and HL-SAN DNase) enabled approximately the same level of human DNA depletion as the known control (MolDNase). Therefore, to test the robustness of the optimized HL-SAN DNase method, the experiment was repeated (with an adjusted volume of HL-SAN buffer required due to limitations of input volume for DNA extraction) against MolDNase with respective DB 1 buffer (Example 3).

Example 3 - Comparison of HL-SAN DNase and MolDNase activity

Here, we tested the robustness of the optimized method selected from Example 2 and compared the activity of HL-SAN DNase and MolDNase with their respective buffers. The volume of HL-SAN buffer which provided the same level of activity between HL-SAN DNase and MolDNase was 180μ1, however, due to the volume input limitation of the MagNAPure (RTM) for DNA purification, the volume of HL-SAN buffer was reduced to 150μ1. Blood cells were lysed by freeze-thawing, spiked with human DNA and HL-SAN DNase or MolDNase was added with their respective buffer, incubated and followed by enzyme heat inactivation. PC was also included, and DNA was extracted from all samples and human qPCR carried out.

Detailed procedure:

1. To lyse blood cells, 2ml of blood was frozen at -70°C and thawed at RT three times

2. Freeze-thawed blood was spiked with human DNA and aliquoted into 4x 250μ1 samples

3. To the HL-SAN DNase sample, 150μ1 of HL-SAN buffer (Example 2) and 4μ1 of HL-SAN DNase was added, mixed by vortexing and incubated at 37°C for 15min

4. To the MolDNase control sample, 50μ1 of buffer DB 1 and 4μ1 of

MolDNase was added, mixed by vortexing and incubated at RT for 15min

5. To the MolDNase control sample and PC PBS was added to increase the sample volume to 400μ1 (the required input volume for the MagNAPure (RTM))

6. DNase activity was stopped by heat killing the enzymes at 65°C for lOmin

7. DNA was extracted and quantified by human qPCR (as described above)

Results:

Table 3 and Figure 3 show that the optimized HL-SAN DNase method out performs the MolDNase control. There is a difference of approximately ACq 2 which equates to an approximate 4-fold reduction in human DNA. Table 3: Human qPCR results of HL-SAN DNase and MolDNase treatment with respective buffers

Conclusion:

Under optimized buffer conditions, HL-SAN DNase can work as, if not more, effectively as MolDNase in blood to deplete human DNA. At this point we continued to work with HL-SAN DNase as our endonuclease of choice and began the process of selecting a suitable cytolysin. Example 4 details the different cytolysins that we initially chose to evaluate for leukocyte cell lysis

ability/efficacy.

Example 4 - Host DNA depletion using Streptolysin O and Alpha hemolysin After identifying HL-SAN DNase as an effective endonuclease for the digestion of DNA, we investigated the potential of cytolysins to target and lyse specific cell types. Here, we evaluated the activity of two membrane pore forming cytolysins, namely streptolysin O {Streptococcus pyogenes) and alpha hemolysin

{Staphylococcus aureus), on leukocyte lysis. Cytolysins were added (individually and in combination) to blood to lyse host cells. Samples were then incubated and released DNA from lysed cells was digested with MolDNase and a DNase inactivation reagent added after further incubation. PC and NTC samples were included and DNA was extracted from all samples and DNA quantified by human qPCR (as detailed above).

Cytolysin purchase information: Streptolysin O

• Cat number no. S5265-25ku

• Lot number 025M4059V

· 25,000-50,000 u/vial

• 0.71mg Solid

• 229577 Units/mg solid

• 4794117 Unts/mg protein

Alpha-hemolysin

• Cat no H9395-5MG

• Lot no 095M4057V

• 28840 Units/mg Solid

• 49647 units/mg protein

Detailed procedure:

1. Streptolysin O and alpha-Hemolysin (0.71 mg (163,000 units) and 5 mg (144,200 units) respectively) was resuspended in 350μ1 of nuclease-free water

2. To sample 1, 50μ1 of Streptolysin O was added to 200μ1 of blood

3. To sample 2, 50μ1 of alpha-hemolysin was added to 200μ1 of blood

4. To sample 3, 50μ1 of Streptolysin O and 50μ1 of alpha-hemolysin was added to 200μ1 of blood

5. All samples were mixed by vortexing and incubated at 37°C with shaking at 400rpm for 30 min

6. After incubation, 150μ1 of HL-SAN buffer was added, followed by 3μ1 of HL-SAN DNase

7. Samples were further incubated at 37°C for 15 min

8. DNase activity was stopped by heat killing the enzymes at 65°C for lOmin

9. To samples 1-3, ΙΟΟμΙ of bacterial lysis buffer was added and to the PC sample 180μ1 of bacterial lysis buffer was added

10. DNA was extracted from all samples and human qPCR used to quantify human DNA (as detailed above)

Results:

When used alone streptolysin O and alpha-hemolysin showed approximately the same leukocyte lysis efficacy (Table 3), providing an approximate 10 ³ fold depletion of DNA. Using both cytolysins in combination (alpha-hemolysin and streptolysin O in combination) on the same blood sample, resulted in improved leukocyte lysis efficiency and improved human DNA depletion with an approximate further 10-fold reduction (ACq 3.3) in human DNA.

Table 4: Human qPCR results after cytolysin treatment

Conclusion:

Here we show that membrane pore forming cytolysins are able to target human cells and enable host DNA depletion. Interestingly, it was the combination of the two cytolysins that produced the greatest human DNA depletion. As we had shown that cytolysins could target human cells and demonstrated that host DNA depletion was possible with this approach, we switched our focus to another member of the cytolysins, namely phospholipase C (PLC) from C. perfringens (which is a cytolysin that breaks down phospholipids in bilayer membranes of eukaryotic cells) (Example 5). Example 5 - Investigation of PLC activity on host cell lysis

As previously mentioned, PLC is a cytolysin produced by C. perfringens and acts by targeting and breaking down phospholipids in the bilayer membrane of eukaryotic cells. We therefore wanted to test PLC for specific host cell lysis and subsequent host DNA digestion using HL-SAN DNase. PLC is a known zinc metallophospholipase and requires the presence of zinc for activity; it was however unknown whether the concentrations of zinc in human blood would be sufficient for PLC to work. Also required for PLC activity are calcium and magnesium ions. With these experiments using blood collected with EDTA preservative, there was a concern that EDTA would chelate the required calcium and metal ions necessary for PLC activity. Therefore, we tested PLC on blood with no preservative, blood containing EDTA preservative and on blood in the presence of a metal ion containing buffer. PLC was added to the various blood sample types and incubated with shaking for host cell lysis. HL-SAN DNase (with

HL-SAN buffer) was then added and incubated for host DNA digestion followed by heat inactivation of HL-SAN DNase. PC and NTC samples were included, and DNA was extracted from all samples followed by human qPCR (as detailed in above).

PLC buffer components:

0.1M ZnCl ₂ and 0.1M MgCl ₂

Detailed procedure:

1. PLC (4mg) was reconstituted in ΙΟΟμΙ of molecular grade water (40μg/μl)

2. Blood was aliquoted into 4x 250μ1

3. To sample 1 (without EDTA preservative) and sample 2 (with EDTA

preservative), 20 μΐ of PLC was added and mixed well by vortexing, followed by incubation at 37°C with shaking at 500rpm for 15min 4. After incubation, 150μ1 of HL-SAN buffer and 4μ1 of HL-SAN DNase was added to samples 1 and 2, mixed well by vortexing and incubated at RT for 15min 5. To sample 3, 150μ1 of HL-SAN buffer and PLC buffer was added followed by 4μ1 of HL-SAN DNase and 20 μΐ of PLC then mixed by vortexing and incubated for 15 min at 37°C without shaking

6. PC was topped up with 150μ1 of PBS (total 400μ1)

7. HL-SAN DNase was inactivated by incubating all samples at 65°C for 10 min

8. DNA was extracted from all samples and human qPCR used to quantify human DNA (as detailed in Section 3)

Results:

There was no improvement in human DNA depletion when PLC was tested on blood with no preservative or with PLC buffer (Table 5 and Figure 5) in fact, the lack of EDTA or addition of PLC buffer reduced the efficacy of depletion. Sample 2 showed the highest level of host DNA depletion with an approximate 100-fold reduction in human DNA compared to the PC (ACq6).

Table 5: Human qPCR results of PLC activity in different sample conditions

Conclusion:

Despite PLC being known to require calcium, magnesium and zinc ions for activity, the addition of buffer containing these ions appeared to decrease the efficiency of PLC to lyse host cells. After concerns that the preservative EDTA would chelate the metal ions required for PLC activity, we observed that PLC worked better in blood samples preserved with EDTA and was less effective in blood without any preservative. All previous experiments were performed in a volume of 200-250μ1 of blood to test the efficiency of PLC and HL-SAN DNase on human DNA depletion. We next wanted to increase the working volume of blood due to the low number of bacterial cells known to be present per millilitre of septic blood (potentially as few as 1 colony forming unit per millilitre)

(Example 6).

Example 6 - Investigation of PLC activity on host DNA depletion and bacterial DNA recovery in an increased volume of blood

The pauci-microbial nature of sepsis means that testing larger volumes of blood increases diagnostic sensitivity. Therefore, we wanted to test the activity of PLC in a larger volume of blood (1ml) and also determine if PLC had any unwanted activity on bacterial cells. Blood was spiked with the most common sepsis causing pathogens (E. coli and S. aureus). Spiked blood was incubated with PLC to enable host cell lysis, followed by the addition of HL-SAN DNase (with HL-SAN buffer) for DNA digestion and the endonuclease was heat inactivated. A PC sample was included and DNA was extracted from all samples, followed by qPCR for human, E. coli and S. aureus DNA (as detailed above).

Detailed procedure:

1. PLC (4mg) was reconstituted in ΙΟΟμΙ of molecular grade water (4C^g^l)

2. Blood spiked with E. coli and S. aureus cultures was aliquoted into lx 1ml and lx 200μ1 samples

3. To 1ml of spiked blood, ΙΟΟμΙ of PLC was added and incubated at 37°C for 20 min with shaking at 500 rpm

4. To 200μ1 of spiked blood, 20μ1 of PLC was added and incubated at 37°C for 20 min with shaking at 500 rpm

5. After incubation, 500μ1 or 150 μΐ of HL-SAN buffer was added to 1ml or 200 μΐ samples respectively, followed by ΙΟμΙ or 3 μΐ of HL-SAN DNase for 1 ml or 200μ1 respectively, mixed briefly by vortexing then incubated at 37°C for 15 min 6. Samples were centrifuged for 10 min at 12,000xg

7. The supernatant was carefully decanted and the pellet was re-suspended in 200μ1 of PBS

8. HL-SAN DNase was inactivated by heat killing at 68°C for 10 min 9. DNA was extracted from all samples and qPCR was used to quantify human, E.coli and S. aureus DNA respectively (as detailed above)

Results:

Increasing the volume of blood resulted in less efficient human DNA depletion (Table 6 and Figure 6 A). There was approximately 4-fold more human DNA remaining in 1ml of blood compared with 200μ1 of blood (ACq2). There was no loss of E. coli between the two volumes, with the 1ml sample showing an approximate 5-fold increase in E. coli DNA (ACq~2.5) as expected (Table 6 and Figure 6B). There was, however, loss of S. aureus DNA in the 200μ1 and 1ml samples, equivalent to approx. 100 fold reduction (ACq~6 in the 200μ1 sample

[lower in the 1ml sample due to the 5 fold increase in volume tested compared to the PC]) (Table 6 and Figure 6C).

Table 6: Human, E. coli and S. aureus qPCR results after PLC and HL-SAN DNase treatment on increased volumes of bacteria spiked blood

Conclusion:

Increasing the volume of blood resulted in less efficient human DNA depletion. Loss of S. aureus DNA was observed suggesting PLC activity on Gram-positive cell walls or a reduction in S. aureus lysis efficiency compared to the PC (possibly due to heat deactivation of DNase). There was no loss of E. coli DNA confirming the Gram-negative bacterial cells were not lysed by PLC. We proceeded to attempt to improve the efficiency of human DNA depletion in 1ml of blood by ensuring effective mixing during incubation with PLC (Example 7). The loss of S. aureus was also investigated using the hypothesis that heat inactivation of HL-

SAN DNase was affecting the cell wall of S. aureus, reducing the efficiency of cell lysis (Example 8).

Example 7 - Investigation of efficient mixing during targeted cell lysis in increased volumes of blood

Firstly, to investigate the loss of PLC efficiency on host cell lysis in 1ml of blood, we investigated the effect of efficient mixing. After the addition of PLC to the bacterial spiked blood, samples were aliquoted in larger volume sample tubes (5ml) and continuously mixed during the incubation period to enhance contact of

PLC with the host cells present in the sample and increase lysis efficiency. HL- SAN DNase (plus HL-SAN buffer) was added to enable host DNA depletion and incubated, followed by heat inactivation. A PC sample was included and DNA was extracted from all samples, followed by qPCR for human, E. coli and S. aureus DNA (as detailed above).

Detailed procedure:

1. PLC (4mg) was reconstituted in ΙΟΟμΙ of molecular grade water (40μg/μl)

2. Blood spiked with E. coli and S. aureus cultures was aliquoted into lx 1ml (in a 5ml tube) and lx 200μ1 samples

3. To 1ml of spiked blood, ΙΟΟμΙ of PLC was added and incubated at 37°C for 20 min with slow mixing using a Hulamixer (RTM)

4. To 200μ1 of spiked blood, 20μ1 of PLC was added and incubated at 37°C for 20 min with shaking at 500 rpm

5. After incubation, 500μ1 or 150 μΐ of HL-SAN buffer was added to 1ml or 200 μΐ samples respectively, followed by ΙΟμΙ or 3 μΐ of HL-SAN DNase for 1 ml or 200μ1 respectively, mixed briefly by vortexing then incubated at 37°C for 15 min

Samples were centrifuged for 10 min at 12,000xg

The supernatant was carefully decanted and the pellet was re-suspended in 200μ1 of PBS

HL-SAN DNase was inactivated by heat killing at 68°C for 10 min DNA was extracted from all samples (including PC) and qPCR was used to quantify human, E.coli and S. aureus DNA respectively (as detailed above) Results:

The introduction of a larger sample tube and slow mixing after the addition of PLC resulted in almost complete removal of human DNA (approximately 1 cell human DNA remaining; a depletion of -2.6 x 10 ⁵ fold (Table 7 and Figure 7) for the 1ml sample and complete removal of human DNA for the 200μ1 sample (a depletion of at least 10 ⁶ fold).

Table 7: Human qPCR results of PLC activity after the addition of efficient mixing during host cell lysis

Conclusion:

By ensuring efficient mixing during host cell lysis the activity of PLC was improved and provided the level of depletion necessary for detecting pathogen sequences in blood by sequencing. However, as described in Example 6, the loss of S. aureus DNA still needed to be investigated (detailed in Example 8). Example 8 - Altered inactivation of HL-SAN DNase to improve Gram-positive bacterial DNA recovery

We hypothesised that heat inactivation of HL-SAN DNase was affecting the cell wall of S. aureus, reducing the efficiency of cell lysis, resulting in low recovery levels of DNA. The aim of this experiment was to try a new method of inactivating HL-SAN DNase in order to improve recovery of S. aureus DNA. Rather than heat inactivation of HL-SAN, we inactivated the DNase by removing the high salt conditions required for its activity. PLC was added to bacterial spiked blood samples, incubated and mixed slowly. HL-SAN DNase (+HL-SAN buffer) was added to enable host DNA depletion and incubated. Samples were centrifuged to pellet the intact bacterial cells and the supernatant containing high salt buffer was removed. A PC sample was included and DNA was extracted from all samples, followed by qPCR for human, E. coli and S. aureus DNA (as detailed above).

Detailed procedure:

1. PLC (4mg) was reconstituted in ΙΟΟμΙ of molecular grade water (4C^g^l)

2. Blood spiked with E. coli and S. aureus cultures was aliquoted into lx 1ml (in a 5ml tube) and lx 200μ1 samples

3. To 1ml of spiked blood, ΙΟΟμΙ of PLC was added and incubated at 37°C for 20 min with slow mixing using a Hulamixer (RTM)

4. To 200μ1 of spiked blood, 20μ1 of PLC was added and incubated at 37°C for 20 min with shaking at 500 rpm

5. After incubation, 500μ1 or 150 μΐ of HL-SAN buffer was added to 1ml or 200 μΐ samples respectively, followed by ΙΟμΙ or 3 μΐ of HL-SAN DNase for 1 ml or 200μ1 respectively, mixed briefly by vortexing then incubated at 37°C for 15 min

6. Samples were centrifuged for 10 min at 12,000xg

7. The supernatant was carefully decanted and the pellet was re-suspended in 1.5ml PBS

8. Prior to DNA extraction, bacterial cells were pelleted by centrifuging at 12000xg for 5min

9. DNA was extracted from all samples (including PC) and qPCR was used to quantify human, E.coli and S. aureus DNA respectively (as detailed above)

Results:

Using buffer exchange rather than heat inactivation on HL-SAN DNase resulted in efficient human DNA depletion with no loss of E. coli or S. aureus DNA (Table 8 and Figure 8). Human DNA depletion was effectively ~ 2.3 x 10 ⁵ fold when using a 1ml sample and (data not shown) at least 10 ⁶ fold when using a

200μ1 sample (no human DNA detected).

Table 8: Human, E. coli and S. aureus qPCR results after altered HL-SAN DNase inactivation

Conclusion:

Introducing a buffer exchange to inactivate HL-SAN DNase instead of heat inactivation, improved the lysis efficiency of S. aureus cells (it is likely that this could also have been achieved by using a more robust lysis method such as bead beating or using an enzyme cocktail). This method alteration enabled efficient S. aureus DNA recovery with no negative effect on E. coli DNA recovery

(previously reported in Example 6) or on human DNA depletion (previously reported in Example 7). Hence an efficient cytolysin human DNA depletion procedure had been developed that did not result in the loss of the microbial component of the sample. In order to confirm the robustness of this procedure we compared it to the commercially available MolYsis (RTM) method and our in- house modified MolYsis (RTM) procedure (Example 9).

Example 9 - Comparison of cytolysin human DNA depletion against MolYsis (RTM) Basic 5 kit and a modified MolYsis (RTM) method

To test the robustness of our newly developed human DNA depletion procedure we compared it to the commercially available MolYsis (RTM) pathogen DNA isolation protocol and an in-house modified MolYsis (RTM) protocol. Our cytolysin human DNA depletion procedure was carried out as per Example 8 using the buffer exchange method rather than heat inactivation of HL-SAN

DNase. The MolYsis (RTM) pathogen DNA isolation protocol was performed as detailed in the manufacturer's instructions. A modified MolYsis (RTM) protocol (developed in house) was also tested which initially removed leukocytes by immunomagnetic separation, followed by MolYsis (RTM) as per the

manufacturer's instructions.

Method 1 (Cytolysin human DNA depletion):

As described in Example 8. Method 2 (MolYsis (RTM)):

MolYsis (RTM) was used as per the manufacturer's instructions.

Method 3 (Modified MolYsis (RTM)):

1. Anti-CD45 coated magnetic beads were re-suspended by gentle mixing then the desired volume of beads (250μ1 per 1ml sample) was aliquoted

2. Beads were washed by re-suspending in 1ml of isolation buffer (25ml Ca ²⁺ ' Mg ²⁺ free PBS, ΙΟΟμΙ 0.5M EDTA and 0.025g BSA)

3. Beads were separated on a magnetic rack and the supernatant was

discarded

4. Beads were re-suspended in 250μ1 of isolation buffer

5. Leukocytes were depleted by adding 250μ1 of washed beads to 1ml of blood and mixed gently at 2-8°C for 30min using a Hulamixer (RTM) 6. Beads were separated on a magnetic rack and the supernatant was transferred to a new sterile tube

7. Intact bacterial cells and any remaining blood cells were pelleted by

centrifugation at 12,000xg for lOmin then the supernatant was discarded

8. The pellet was re-suspended in 1ml PBS

9. Samples were further processed using the MolYsis (RTM) protocol

according to the manufacturer' s instructions

DNA was extracted from all samples (including PC) and qPCR was used to quantify human, E.coli and S. aureus DNA respectively for all methods (as detailed above)

Results:

When comparing our human DNA depletion method to commercially available MolYsis (RTM) we observed approximately 10 ⁴ -fold more human DNA depletion (ACql2) and comparable levels of bacterial DNA recovery (Table 9 and Figure 9). Our modified MolYsis (RTM) protocol also showed an approximate 10 ⁴ -fold reduction in human DNA (ACql2) compared to MolYsis (RTM).

Table 9: Human, E. coli and S. aureus qPCR results for method comparison

Conclusion:

In comparison to the commercially available MolYsis (RTM) kit, our human DNA depletion method was more efficient at human DNA depletion (showing ~ 9.3 x 10 ⁴ fold depletion of human DNA). Only our modified MolYsis (RTM) protocol showed the same level of efficiency compared to our cytolysin human DNA depletion method. This demonstrates that the leading commercially available host depletion kit does not provide sufficient host cell/DNA depletion to enable efficient pathogen DNA detection by sequencing.

Overview:

In conclusion, we have developed a rapid pathogen identification procedure which utilizes the properties of cytolysins (PLC) and endonucleases (HL-SAN DNase) to specifically target and lyse host cells present in clinical samples (i.e. blood), followed by DNA digestion. This procedure is a pre-step to enable sufficient pathogen DNA extraction for NGS. As blood represents the most complex clinical sample matrix type with extremely high human to bacterial cell ratios, we predict that the clinical sample type will be easily interchangeable without affecting the levels of human DNA depletion.

After a number of methodology alterations, the finalised procedure is detailed below.

Initially optimised human DNA depletion method

PLC solution: 4mg in ΙΟΟμΙ nuclease free water

HL-SAN buffer: lOmM Tris HCL, lOOmM Magnesium and lM NaCl pH8.5 in nuclease free water

ΙΟΟμΙ PLC solution was added to 1ml blood

I

Incubated at 37°C with gentle mixing for 20min

I

500μ1 HL-SAN buffer, ΙΟμΙ HL-SAN DNase was added and mixed by vortexing, then incubated at 37°C for 15min I

Bacterial cells were pelleted at 12,000xg for lOmin

I

Supernatant was discarded

I

Bacterial cell pellet was resuspended in 1.5ml PBS

I

Pellet bacterial cells at 12,000xg for 5mins and remove supernatant

I

Proceeded to DNA extraction of choice

[Total time: 50min.]

DNA extraction

Bacterial cell pellet was resuspended in 350μ1 bacterial lysis buffer and vortexed

I

30μ1 enzyme cocktail (lysozyme, mutanolysis and lysostaphin - lyticase optional) was added and incubated at 37°C for 15min at lOOOrpm

I

20 μΐ proteinase K was added

I

Mixed by vortexing

I

Incubated at 65°C for 5min

I

Proceed to MagNAPure (RTM) (Roche) for DNA extraction

[Total time: 45min.]

[Therefore current protocol turnaround time approximately 90min.] Example 10 - verification of methodology for fungal enrichment

10.1 : The protocol above was altered slightly to focus on fungal enrichment and the final protocol was carried out to verify bacterial enrichment. The protocol was tested using -200 E. coli cells. Blood was spiked with -200 E. coli cells and was processed as detailed in section 10.2.

10.2: Amended protocol ("Enrichment" procedure): 1 PLC was added (0.8 mg/20 μΐ) to the blood sample (200 μΐ), vortexed and incubated at 37 °C for 15 min at 1000 RPM in a heatblock.

2 HL-SAN buffer (5M NaCl and 1 OOmM MgCl ₂ ) was added at a 1 : 1 volume ratio (200 μΐ) with 10 μΐ HL-SAN DNase, vortexed and incubated at 37 °C for 15 min at 1000 RPM in a heat block.

3 PBS was added to a total volume of 2 ml (1.5 ml).

4 Cells were pelleted by centrifugation at 12,000 xg for 10 min and the supernatant was discarded.

5 The cell pellet was resuspended in 1.5 ml PBS.

6 Cells were pelleted again by centrifugation at 12,000 xg for 10 min and the supernatant was discarded.

7 To any test samples; 350 μΐ bacterial lysis buffer, 20 μΐ enzyme cocktail (6 μΐ mutanolysin 25 ku/ml, 5 μΐ lysozyme 10 mg/ml, 4 μΐ lyticase 10 ku/ml, 3 μΐ lysostaphin 4 ku/ml, 2 μΐ chitinase 50 u/ml) and 5 μΐ RNase A was added.

8 All samples were incubated at 37 °C for 15 min at 1000 RPM in a heat block.

9 To all samples, 20 μΐ proteinase K was added and incubated at 65 °C for

10 min in a heat block.

10 Total nucleic acid was extracted using the MagnaPure (RTM) Compact automated machine using the DNA_bacteria_V3_2 protocol.

11 Host DNA/RNA depletion and fungal DNA enrichment was determined via qPCR or RT-qPCR. Results:

After plate counts it was identified that 200 μΐ of blood was spiked with ~110 E. coli cells. This resulted in ~10 ⁵ fold depletion of human DNA and no loss of E. coli DNA (Tables lO. la/b).

Table 10.1a Human DNA qPCR results for -110 E. coli cells spiked blood with and without fungal/bacterial enrichment.

Table 10.1b E. coli DNA qPCR results for -110 E. coli cells spiked blood with and without fungal/bacterial enrichment.

Whole blood was spiked with -1000 C. albicans cells and two samples were processed as detailed in section 10.2. After the enrichment protocol there was between -10 ⁴ and -10 ⁵ fold depletion of human DNA and no loss of C. albicans

DNA (Tables 10.2a/b). Table 10.2a Human DNA qPCR results in duplicate for <1000 C. albicans cells spiked blood with and without bacterial/fungal enrichment.

Sample ID Human Average Human Average ACq qPCR assay (Cq) (Cq) against PC

PC blood spiked 24.37

(PC 1)

PC blood spiked 24.32 24.3

(PC 2)

Blood spiked Undetectable 14.9

Enriched (>40)

(Sample 1) 39.2

Blood spiked 38.33

Enriched

(Sample 2)

Table 10.2b C. albicans DNA qPCR results in duplicate for <1000 C. albicans cells spiked blood with and without bacterial/fungal enrichment.

Sample ID C. albicans Average C. Average ACq qPCR assay (Cq) albicans against PC

(Cq)

PC blood spiked 33.91

(PC 1)

PC blood spiked 33.28 33.6

(PC 2)

Blood spiked 30.81

Enriched 2.3

(Sample 1) 31.3

Blood spiked 31.81

Enriched

(Sample 2) Whole blood was then spiked with -200 C. albicans cells and was processed as detailed in section 10.2. After plate counts of C. albicans on sabouraud agar, it was identified that 200 μΐ of blood was spiked with -60 C. albicans cells. After the enrichment protocol this resulted in -10 ⁵ fold depletion of human DNA and no loss of C. albicans DNA (Tables 10.3a/b).

Table 10.3a Human DNA qPCR results in for ~60 C. albicans cells spiked blood with and without bacterial/fungal enrichment.

Table 10.3b C. albicans DNA qPCR results in for -60 C. albicans cells spiked blood with and without bacterial/fungal enrichment.

Using the A. niger bioball known to be -10 cfu/ml, serial dilutions were made to

-10 ⁴ and -10 ³ . Both samples were processed as described in section 10.2. After the enrichment protocol this resulted in ~10 ⁵ fold depletion of human DNA and no loss of niger DNA (Tables 10.4a-b/10.5a-b).

Table 10.4a Human DNA qPCR results for -200 A. niger cells (10 ³ dilution) spiked blood with and without bacterial/fungal enrichment.

Table 10.4b A. niger DNA qPCR results for -200 A. niger cells (10 ³ dilution) spiked blood with and without bacterial/fungal enrichment.

Sample ID A. niger qPCR ACq against PC

assay (Cq)

PC blood spiked 39.21

(PC 1)

0.79

Blood spiked 40

Enriched

(Sample 1) Table 10.5a Human DNA qPCR results for -2,000 A. niger cells (10 ⁴ dilution) spiked blood with and without bacterial/fungal enrichment.

Table 10.5b A. niger DNA qPCR results for -2,000 A. niger cells (10 ⁴ dilution) spiked blood with and without bacterial/fungal enrichment.

* Cq value suggests <10 cell (<100 cells in total input)

Conclusion: Using the protocol detailed in section 10.2, there is ~10 ⁵ fold human DNA depletion with no loss of bacterial or fungal DNA. Example 11 - verification of methodology for virus and phage enrichment 11.1 : Protocol for viral enrichment in plasma

1. Whole blood was spiked with viral particles (max 200 μΐ per sample).

2. Samples were centrifuged at 20,000 xg for 5 min.

3. Supernatant was retained and used for the protocol (effectively working in plasma) after being aliquoted into equal volumes (max 200 μΐ).

4. 20 μΐ of PLC (0.8 mg) was added to each test sample and incubated at 37 °C for 15 min with shaking at 1000 RPM in a heat-block.

5. 200 μΐ of HL-SAN buffer (5 M NaCl and 100 mM MgCl ₂ ) and 10 μΐ HL- SAN was added, incubated at 37 °C for 15 min with shaking at 1000 RPM in a heat-block.

6. 20 μΐ proteinase K was added to all samples and incubated at 65 °C for lOmin.

7. Total nucleic acid was extracted using the MagnaPure (RTM) Compact automated machine using the DNA_bacteria_V3_2 protocol.

8. Host DNA/RNA depletion and viral DNA/RNA enrichment was

determined via qPCR or RT-qPCR.

11.2: Protocol for viral enrichment in blood

1. Whole blood was spiked with viral particles (max 200 μΐ per sample).

2. 20 μΐ of PLC (0.8 mg) was added to each test sample and incubated at 37 °C for 15 min with shaking at 1000 RPM in a heat-block.

3. 200 μΐ of HL-SAN buffer (5 M NaCl and 100 mM MgCl ₂ ) and 10 μΐ HL- SAN was added, incubated at 37 °C for 15 min with shaking at 1000 RPM in a heat-block.

4. Test samples were centrifuged at 20,000 xg for 5 min and the supernatant retained.

5. 20 μΐ proteinase K was added to all samples and incubated at 65 °C for 10 min. 6. Total nucleic acid was extracted using the MagnaPure (RTM) Compact automated machine using the DNA_Bacteria_V3_2 protocol.

7. Host DNA/RNA depletion and viral DNA/RNA enrichment was

determined via qPCR or RT-qPCR.

Once the protocols described in sections 11.1 and 11.2 were established, samples were run in triplicate to access the reproducibility of the protocols (a second blood protocol was also tested at this stage which was the same as section 11.2 with an additional centrifugation step after step 4).

Results:

In total, each 200 μΐ blood sample was spiked with 10,000 IU HIV and 350 IU HBV. For this experiment, all three enrichment protocols were tested in triplicate (as previously described), After the viral enrichment protocols in blood there was consistently ~10 ⁴ fold depletion in human DNA and human DNA was

undetectable after enrichment when working in plasma (Tables 1 l . la/b).

There was no loss of HBV viral DNA target in blood and plasma, although it should be noted that the number of HBV cells in the PCR reactions was -35 and so Cq values were close to the limit of detection for the qPCR assay used (Tables 11.2a/b). With regards RNA viral targets, there was no loss of HIV in blood and plasma (Tables 11.3a/b).

Table 11.1a Human DNA qPCR results in triplicate for spiked blood with and without viral enrichment.

Sample ID Human Average Human Average ACq qPCR assay (Cq) (Cq) against PC

PC blood spiked 24.34

1

(PC #1)

PC blood spiked 25.04 24.81

2

(PC #2)

PC blood spiked 25.06

3

(PC #3)

Blood spiked 37.32

Enriched 1

1

(T_l #1) 12.73

Blood spiked 37.68 37.54 (10 ^A 4) Enriched 1

2

(T_l #2)

Blood spiked 37.91

Enriched 1

3

(T_l #3) Blood spiked 37.94

Enriched 2

1

(T_2 #1) 38.16 13.35

Blood spiked 38.64 (10 ^A 4) Enriched 2

2

(T_2 #2)

Blood spiked 37.91

Enriched 2

3

(T_2 #3)

Table 11.1b Human DNA qPCR results in triplicate for spiked plasma with and without viral enrichment.

Sample ID Human Average ACq

qPCR assay (Cq) against PC

PC plasma spiked 34.64

1

(PC_SN #1)

PC plasma spiked 33.45

2

(PC_SN #2)

PC plasma spiked 33.81

3 Undetectable

(PC_SN #3)

Plasma spiked Undetectable

Enriched

1

(T_SN #1)

Plasma spiked Undetectable

Enriched

2

(T_SN #2)

Plasma spiked Undetectable

Enriched

3

(T_SN #3) Table 11.2a HBV DNA qPCR results in triplicate for spiked blood with and without viral enrichment.

Sample ID HBV Average HBV Average ACq qPCR assay (Cq) (Cq) against PC

PC blood spiked 38.02

1

(PC #1)

PC blood spiked 36.95 37.9

2

(PC #2)

PC blood spiked 38.76

3

(PC #3)

Blood spiked 39.12

Enriched 1

1

(T_l #1) 38 0.1

Blood spiked 37.99

Enriched 1

2

(T_l #2)

Blood spiked 36.81

Enriched 1

3

(T_l #3) Blood spiked 37.37

Enriched 2

1

(T_2 #1) 37.4 0.5

Blood spiked 37.47

Enriched 2

2

(T_2 #2)

Blood spiked Undetectable

Enriched 2

3

(T_2 #3)

Table 11.2b HBV DNA qPCR results in triplicate for spiked plasma with and without viral enrichment.

Sample ID HBV Average ACq

qPCR assay (Cq) against PC

PC plasma spiked 37.62

1

(PC_SN #1)

PC plasma spiked 36.92

2

(PC_SN #2)

PC plasma spiked 36.95

3 0.02

(PC_SN #3)

Plasma spiked 37.22

Enriched

1

(T_SN #1)

Plasma spiked Undetectable

Enriched

2

(T_SN #2)

Plasma spiked Undetectable

Enriched

3

(T_SN #3) Table 11.3a HIV RNA RT-qPCR results in triplicate for spiked blood with and without viral enrichment.

Sample ID HIV Average HIV Average ACq qPCR assay (Cq) (Cq) against PC

PC blood spiked 32.76

1

(PC #1)

PC blood spiked 33.60 33.2

2

(PC #2)

PC blood spiked 33.14

3

(PC #3)

Blood spiked 33.33

Enriched 1

1

(T_l #1) 33.5 0.3

Blood spiked 34.02

Enriched 1

2

(T_l #2)

Blood spiked 33.08

Enriched 1

3

(T_l #3) Blood spiked 33.63

Enriched 2

1

(T_2 #1) 33.7 0.5

Blood spiked 33.75

Enriched 2

2

(T_2 #2)

Blood spiked 33.65

Enriched 2

3

(T_2 #3)

Table 11.3b HIV RNA RT-qPCR results in triplicate for spiked plasma with and without viral enrichment.

Sample ID HIV Average HIV Average ACq qPCR assay (Cq) (Cq) against PC

PC plasma spiked 34.44

1

(PC_SN #1)

PC plasma spiked 33.75 34.6

2

(PC_SN #2)

PC plasma spiked 35.64

3

(PC_SN #3)

Plasma spiked 35.66

Enriched

1 0.4

(T_SN #1)

Plasma spiked 35.00 34.9

Enriched

2

(T_SN #2)

Plasma spiked 34.03

Enriched

3

(T_SN #3) Next, for phage testing; in total, each 200 μΐ blood sample was spiked with either 10 ⁴ , 10 ⁵ , 10 ⁶ or 10 ⁷ phage. After the viral enrichment protocol in plasma (section 11.1) there was consistently ~10 ³ fold depletion in human DNA with no loss of phage target (Tables 11.4a/b).

Table 11.4a Human DNA qPCR results for spiked blood with and without viral enrichment.

Sample ID Human qPCR Average ACq

assay (Cq) against PC

PC blood spiked 28.01

10 ⁴ 11.99

Blood spiked 40

Enriched

10 ⁴

PC blood spiked 28.68

10 ⁵ 11.32

Blood spiked 40

Enriched

10 ⁵

PC blood spiked 28.72

10 ⁶ 11.28

Blood spiked 40

Enriched

10 ⁶

PC blood spiked 28.43

10 ⁷

Blood spiked 37.95 9.52

Enriched

10 ⁷ Table 11.4b Phage DNA qPCR results for spiked blood with and without viral enrichment.

Conclusion:

Here we described a complete protocol for the depletion of host DNA and enrichment of viral (both DNA and RNA) and phage (DNA). Two methods have been developed (one working in plasma; section 1 1.1, and one working in blood; section 11.2), and both provide human DNA depletion (~10 ⁴ fold depletion in blood to undetectable in plasma). There is no loss of viral and phage DNA targets or viral HIV RNA target. Example 12 - altering the cytolysin (blood samples)

For all testing with other cytolysins, 200 μΐ of blood was used following the protocol set out in section 10.2. The only alteration was the addition of different volumes/concentrations in place of PLC, i.e. no optimization was carried out.

Phospholipase D (PLD) from Streptomyces

PLD was purchased from Sigma-Aldrich (RTM) (P0065-25KU) with a stock made to 50KU/ml; varying volumes of PLD were used (2, 5 and 8 μΐ). Human

DNA was depleted <10 ² fold (Table 12.1a) with no loss of bacterial or fungal targets (Tables 12.1b,c,d).

Table 12.1a Human DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using PLD.

Sample ID Human qPCR Average ACq

assay (Cq) against PC

PC blood 1 23.07

Unenriched

3.68

Blood 1 26.75

Enriched 2μ1

PC blood 2 23.05

Unenriched

5.76

Blood 2 28.81

Enriched 5μ1

PC blood 3 23.28

Unenriched

Blood 3 27.04 3.76

Enriched 8μ1 Table 12.1b E. coli DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using PLD.

Sample ID E. coli qPCR assay Average ACq

(Cq) against PC

PC blood 1 25.98

Unenriched

1.02

Blood 1 24.96

Enriched 2μ1

PC blood 2 25.29

Unenriched 0.2

Blood 2 25.09

Enriched 5μ1

PC blood 3 27.32

Unenriched

Blood 3 26.77 0.55

Enriched 8μ1

Table 12.1c S. aureus DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using PLD.

Sample ID S aureus qPCR Average ACq

assay (Cq) against PC

PC blood 1 24.01

Unenriched

0.69

Blood 1 23.32

Enriched 2μ1

PC blood 2 23.20

Unenriched

0.78

Blood 2 23.98

Enriched 5μ1

PC blood 3 23.06

Unenriched

Blood 3 22.73 0.33

Enriched 8μ1

Table 12.1d C. albicans DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using PLD.

Sample ID C albicans qPCR Average ACq

assay (Cq) against PC

PC blood 1 29.55

Unenriched

0.53

Blood 1 29.02

Enriched 2μ1

PC blood 2 29.58

Unenriched

0.35

Blood 2 29.93

Enriched 5μ1

PC blood 3 29.91

Unenriched

Blood 3 29.76 0.15

Enriched 8μ1

Sphingomyelinase from S. aureus

Sphingomyelinase was purchased from Sigma-Aldrich (RTM) (S8633-25UN) in solution and varying volumes were used (2, 5 and 8 μΐ). Human DNA was depleted <10 ² fold (Table 12.2a) with no loss of bacterial or fungal targets (Tables

12.2b,c,d).

Table 12.2a Human DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using sphingomyelinase.

Sample ID Human qPCR Average ACq

assay (Cq) against PC

PC blood 1 23.07

Unenriched

4.57

Blood 1 27.64

Enriched 2μ1

PC blood 2 23.05

Unenriched

7.53

Blood 2 30.58

Enriched 5μ1

PC blood 3 23.28

Unenriched

Blood 3 28.74 5.46

Enriched 8μ1 Table 12.2b E. coli DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using sphingomyelinase.

Sample ID E. coli qPCR assay Average ACq

(Cq) against PC

PC blood 1 25.98

Unenriched

1.61

Blood 1 24.67

Enriched 2μ1

PC blood 2 25.29

Unenriched

Blood 2 25.26 0.03

Enriched 5μ1

PC blood 3 27.32

Unenriched

Blood 3 26.67 0.65

Enriched 8μ1

Table 12.2c S.aureus DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using sphingomyelinase.

Sample ID S aureus qPCR Average ACq

assay (Cq) against PC

PC blood 1 24.01

Unenriched

1.36

Blood 1 22.65

Enriched 2μ1

PC blood 2 23.20

Unenriched

0.92

Blood 2 24.12

Enriched 5μ1

PC blood 3 23.06

Unenriched

Blood 3 22.66 0.73

Enriched 8μ1

Table 12.2d C. albicans DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using sphingomyelinase.

Sample ID C albicans qPCR Average ACq

assay (Cq) against PC

PC blood 1 29.55

Unenriched

1.73

Blood 1 27.82

Enriched 2μ1

PC blood 2 29.58

Unenriched

0.11

Blood 2 29.69

Enriched 5μ1

PC blood 3 29.91

Unenriched

Blood 3 28.99 0.92

Enriched 8μ1

Alpha hemolysin from S. aureus

Alpha hemolysin was purchased from Sigma-Aldrich (RTM) (H9395-5MG) and added at 0.01, 0.08 or 0.8mg in 20μ1 water. Human DNA was depleted <10 ² fold (Table 12.3a) with no loss of bacterial or fungal targets (Tables 12.3b,c,d).

Table 12.3a Human DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using alpha hemolysin.

Sample ID Human qPCR Average ACq

assay (Cq) against PC

PC blood 1 23.06

Unenriched 1.45

Blood 1 24.48

Enriched O.Olmg

PC blood 2 23.28

Unenriched

4.38

Blood 2 27.63

Enriched 0.08mg

PC blood 3 23.28

Unenriched

Blood 3 27.22 3.94

Enriched 0.8mg Table 12.3b E. coli DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using alpha hemolysin.

Sample ID E. coli qPCR assay Average ACq

(Cq) against PC

PC blood 1 26.96

Unenriched

0.59

Blood 1 26.37

Enriched O.Olmg

PC blood 2 27.32

Unenriched

0.11

Blood 2 27.21

Enriched 0.08mg

PC blood 3 27.32

Unenriched

Blood 3 27.34 0.02

Enriched 0.8mg

Table 12.3c S. aureus DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using alpha hemolysin.

Sample ID S aureus qPCR Average ACq

assay (Cq) against PC

PC blood 1 22.71

Unenriched

0.41

Blood 1 23.12

Enriched O.Olmg

PC blood 2 23.06

Unenriched

Blood 2 22.73 0.3

Enriched 0.08mg

PC blood 3 23.06

Unenriched

Blood 3 23.12 0.06

Enriched 0.8mg

Table 12.3d C. albicans DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using alpha hemolysin.

Sample ID C albicans qPCR Average ACq

assay (Cq) against PC

PC blood 1 28.57

Unenriched

0.41

Blood 1 28.16

Enriched O.Olmg

PC blood 2 29.91

Unenriched

1.75

Blood 2 28.16

Enriched 0.08mg

PC blood 3 29.91

Unenriched

Blood 3 29.81 0.1

Enriched 0.8mg

Streptolysin O from S. pyogenes

Streptolysin O was purchased from Sigma- Aldrich (RTM) (S5265-25KU) and added at 0.08 or 0.8mg in 20μ1 water. Human DNA was depleted 10 fold (Table 12.4a) with no loss of bacterial or fungal targets (Tables 12.4b,c,d).

Table 12.4a Human DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using streptolysin O.

Sample ID Human qPCR Average ACq

assay (Cq) against PC

PC blood 1 23.28

Unenriched

2.87

Blood 1 26.15

Enriched 0.08mg

PC blood 2 23.28

Unenriched

Blood 2 26.18 2.9

Enriched 0.8mg

Table 12.4b E. coli DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using streptolysin O.

Table 12.4c S. aureus DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using streptolysin O.

Sample ID S aureus qPCR Average ACq

assay (Cq) against PC

PC blood 1 23.06

Unenriched

0.38

Blood 1 22.68

Enriched 0.08mg

PC blood 2 23.06

Unenriched

Blood 2 22.78 0.28

Enriched 0.8mg Table 12.4d C. albicans DNA qPCR results for spiked blood with and without bacterial/fungal enrichment using streptolysin O.

Conclusion:

All cytolysins tested showed effective human DNA depletion and no bacterial fungal DNA loss.

Example 13 - verification of methodology for other clinical sample types

Using the established protocol detailed in section 10.2, the initial 200μ1 of blood was replaced with 200μ1 of sputum, sonicated tissue or urine to verify the depletion method works effectively in other clinical sample types.

Clinical sputum samples

Human DNA was depleted up to 10 ⁴ fold (Table 13.1a) with no loss of bacteria (Tables 13.1b/c) in clinical sputum samples.

Table 13.1a Human DNA qPCR results for clinical sputum with and without fungal/bacterial enrichment.

Sample ID Human qPCR Average ACq

assay (Cq) against PC

PC sputum 1 19.81

Unenriched

8.08

Sputum 1 27.89

Enriched

PC sputum 2 22.10

Unenriched

12.31

Sputum 2 34.41

Enriched Table 13.1b 16S rRNA gene fragment (V3-V4) qPCR results for clinical sputum with and without fungal/bacterial enrichment.

Table 13.1c S. aureus DNA qPCR results for clinical sputum with and without fungal/bacterial enrichment.

Sample ID S aureus qPCR Average ACq

assay (Cq) against PC

PC sputum 2

Unenriched 22.29

(suspected S

aureus) 0.87

Sputum 2

Enriched 22.96

(suspected S

aureus) Peri-prosthetic tissue samples

Peri-prosthetic tissue sample biopsies spiked with Staphylococcus epidermidis cells (15TB0821), with <10 ⁵ fold human DNA depletion (Table 13.2a) and no loss of bacterial target (Table 13.2b).

Table 13.2a Human DNA qPCR results for per-prosthetic spiked tissue samples with and without fungal/bacterial enrichment.

Table 13.2b S. epidermidis DNA qPCR results for peri-prosthetic spiked tissue samples with and without fungal/bacterial enrichment.

Sample ID S epidermidis qPCR ACq against PC

assay (Cq)

PC tissue 100 cells 37.25

Unenriched

Tissue 100 cells 35.26 1.99

Enriched Clinical urine samples

Human DNA was depleted <10 ⁴ fold (Table 13.3 a) with no loss of bacteria (Tables 13.3b/c) in clinical sputum samples.

Table 13.3a Human DNA qPCR results for clinical urine with and without fungal/bacterial enrichment.

Sample ID Human qPCR Average ACq

assay (Cq) against PC

PC urine 1 24.01

Unenriched

10.99

Urine 1 35

Enriched

PC urine 2 31.26

Unenriched

3.74

Urine 2 35

Enriched

PC urine 3 24.98

Unenriched

Urine 3 35 10.32

Enriched

Table 13.3b 16S rRNA gene fragment (V3-V4) qPCR results for clinical urine with and without fungal/bacterial enrichment.

Table 13.3c E. coli DNA qPCR results for clinical urine with and without fungal/bacterial enrichment.

Conclusion: All clinical sample types tested showed host DNA depletion with no loss of bacterial DNA. Example 14 - host RNA depletion (HL-SAN RNase activity)

There was >10 ² fold host RNA depletion using the viral blood protocol (section 11.2 and Table 14.1a). Using the viral plasma protocol detailed in section 11.1, showed >10 ² fold depletion of host RNA (Table 14.1b and 14.2a) with no loss of

HIV target (Table 14.2b).

Table 14.1a Human RNA RT-qPCR results in duplicate for non-spiked blood with and without viral enrichment (host RNA depletion).

Sample ID Human RNA Average ACq

qPCR assay (Cq) against PC

Unenriched blood 24.72

non-spiked

1 8.53

Enriched blood 33.25

non-spiked

1

Unenriched blood 32.49

non-spiked

2 5.9

Enriched blood 38.39

non-spiked

2

Table 14.1b Human RNA RT-qPCR results in duplicate for non- plasma with and without viral enrichment (host RNA depletion).

Sample ID Human RNA Average ACq qPCR assay (Cq) against PC

Unenriched 36.26

plasma unspiked

1 8.74

Enriched plasma Undetectable

Unspiked

1

Unenriched 34.44

plasma unspiked

2 10.56

Enriched plasma Undetectable

Unspiked

2

Table 14.2a Human RNA RT-qPCR results in duplicate for spiked plasma with and without viral enrichment (host RNA depletion).

Sample ID Human RNA Average ACq

qPCR assay (Cq) against PC

Unenriched plasma 36.35

spiked

1 8.65

Enriched plasma Undetectable

spiked

1

Unenriched plasma 30.87

spiked

2

Enriched plasma 34.15 3.28

spiked

2

Table 14.2b HIV RNA RT-qPCR results in duplicate for spiked plasma with and without viral enrichment (host RNA depletion).

Conclusion:

Due to the variability of starting host RNA, it was established that HL-SAN RNase activity provided the greatest host RNA depletion with no loss of viral RNA target and therefore no alterations to the enrichment protocol (detailed in section 11.1) was necessary. Human RNA was typically not detectable in plasma post depletion using this method.

Example 15 - removal of human DNA without nuclease

Propidium monoazide (PMA) to remove human DNA An altered method from that described in section 10.2 was needed to enable the activation of PMA by light. After PLC treatment, the sample was centrifuged at 12,000xg for 5min and resuspended in 1.5ml of PBS. PMA was added at a final concentration of 50μΜ and incubated in the dark with occasional shaking for 5min. The sample was then placed in a photolysis device for 15min exposure to blue light, the protocol in section 10.2 was then followed from step 6. Human

DNA was depleted <10 ² fold (Table 15.1) with no loss of bacterial target DNA (Table 15.2).

Table 15.1 Human DNA qPCR results for spiked blood samples with and without fungal/bacterial enrichment using PMA to remove human DNA.

Sample ID Human qPCR ACq against PC

assay (Cq)

PC blood 22.90

Unenriched

Blood PMA #1 27.62 4.72

Enriched

Blood PMA #2 28.47 5.57

Enriched

Table 15.2 E. coli DNA qPCR results for spiked blood samples with and without fungal/bacterial enrichment using PMA to remove human DNA.

Conclusion:

Using PMA to remove human DNA after PLC treatment showed human DNA depletion and no loss of bacterial target DNA

Example 16 - revised protocol for 1ml blood sample

1 PLC was added (4 mg/100 μΐ) to the blood sample (1 ml in a 5 ml bijou tube), vortexed and incubated at 37 °C for 3 min in a water bath followed by 38 °C for 20 min with slow mixing at 15rpm in a hulamixer (RTM).

2 Sample was transferred to a 2 ml tube and 500 μΐ of HL-SAN buffer (5M NaCl and lOOmM MgCl ₂ ) was added and incubated 37 °C for 15 min in a heatblock at 1000 RPM.

3 Cells were pelleted by centrifugation at 8,000 xg for 5 min.

4 The cell pellet was resuspended in 200 μΐ PBS

5 HL-SAN buffer was added at a 1 : 1 volume ratio (200 μΐ) with 10 μΐ HL- SAN DNase, vortexed and incubated at 37 °C for 15 min at 1000 RPM in a heat block.

6 PBS was added to a total volume of 2 ml (1.5 ml).

7 Cells were pelleted by centrifugation at 12,000 xg for 10 min and the supernatant was discarded.

8 The cell pellet was resuspended in 1.5 ml PBS.

9 Cells were pelleted again by centrifugation at 12,000 xg for 10 min and the supernatant was discarded.

10 To any test samples; 350 μΐ bacterial lysis buffer, 20 μΐ enzyme cocktail (6 μΐ mutanolysin 25 ku/ml, 5 μΐ lysozyme 10 mg/ml, 4 μΐ lyticase 10 ku/ml, 3 μΐ lysostaphin 4 ku/ml, 2 μΐ chitinase 50 u/ml) and 5 μΐ RNase A was added.

11 All samples were incubated at 37 °C for 15 min at 1000 RPM in a heat block.

12 To all samples, 20 μΐ proteinase K was added and incubated at 65 °C for 10 min in a heat block.

13 Total nucleic acid was extracted using the MagnaPure (RTM) Compact automated machine using the DNA_bacteria_V3_2 protocol.

Changes to the 200μ1 protocol in section 10.2 to increase the starting volume to lml are described above. This gave >10 ⁶ fold depletion of human DNA (Table 16. la) with no loss of bacterial or fungal target DNA (Tables 16. lb,c,d). Table 16.1a Human DNA qPCR results for 1ml spiked blood with and without fungal/bacterial enrichment.

Table 16.1b E. coli DNA qPCR results for 1ml spiked blood with and without fungal/bacterial enrichment.

Table 16.1c S. aureus DNA qPCR results for 1ml spiked blood with and without fungal/bacterial enrichment.

Sample ID S aureus qPCR ACq against PC

assay (Cq)

PC blood 37.63

Unenriched

3.72

Blood 1ml 33.91

Enriched Table 16. Id C. albicans DNA qPCR results for 1ml spiked blood with and without fungal/bacterial enrichment.

Conclusion:

A slightly altered method was developed to enable fungal enrichment when using lml blood and this resulted in ~10 ⁶ fold depletion of human DNA with no loss of bacteria or fungi target DNA. Greater sample volumes (>lml) could also be used.

This method can seemingly be used on any sample type where the host cells have a phospholipid membrane e.g. clinical samples (infectious disease diagnosis) or animal samples (food safety and veterinary medicine/diagnosis).

Example 17 - NGS after depletion method

Additional methodology

After the depletion protocol detailed in section 10.2, 4μ1 DNA was processed using REPLI-g single cell kit (Qiagen 150343) for whole genome amplification

(WGA). The manufacturer's instructions were followed with the amplification time reduced to lhr 30min. WGA sample (17μ1) was debranched using T7 endonuclease I (NEB M0302S) according to the manufacturer's instructions. MinlON library preparation used the rapid low input by PCR barcoding kit (ONT SQK-RLBOOl) as per the manufacturer's guideline with the following alterations:

• 2.5μ1 FRM with 7.5μ1 template DNA (~140ng)

• 40 μΐ nuclease-free water, 50μ1 Long Amp Taq 2x, 2μ1 RLB

• PCR: [95°C 3min]xl, [95°C 15s, 56°C 15s, 65°C 4min]x20, [65°C

4min]x20, [65°C 6min]xl

The SpotON R9.4 MinlON flowcell was prepared and loaded according to the manufacturer's instructions.

Bioinformatics data analysis: reads were aligned to the C. albicans reference genome (SC5314 NC_003977.2) using minimap2. Genome coverage and number of aligned reads were identified using samtools and qualimap. Percentage reads are given as those which aligned to the reference genome out of the total number of reads.

Results

~300cfu/ml Candida albicans at ~15Mb genome = 4.5pg of DNA

Average concentration of human DNA in 1ml blood = 33μg of DNA

Therefore before enrichment the ratio of human: Candida DNA is -10

From the sequencing data presented below, C albicans reads are 1% of the total (1.3x genome coverage) therefore assuming all other reads are human = 100: 1 (human : Candida) Ratio of human .Candida DNA before depletion = 10 : 1

Ratio of human .Candida DNA after depletion = 100: 1

This is the equivalent of 10 ⁵ fold depletion.

Table 17 C albicans genome alignment from single-plex MinlON run (input ~300cfu/ml).

C. albicans genome coverage plot after C. albicans single-plex MinlON sequencing is shown in Figure 10.