Method for whole genome sequencing (WGS) of microbes

Abstract

The present disclosure provides method of whole genome sequencing of microorganisms such as Gram-positive bacteria, Gram-negative bacteria, viruses, and fungi. The various methods comprising steps of isolating nucleic acid from the microorganism and preparing a library of the isolated nucleic acid. Preferably, the methods of whole genome sequencing described herein are automated to provide a robust and efficient sequencing product for various utilities.

Description

78090-429588-1-METHOD FOR WHOLE GENOME SEQUENCING (WGS) OF MICROBESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No. 63 / 685,044, filed on August 20, 2024. the entire disclosures of which is incorporated herein by reference.BACKGROUND AND SUMMARY

[0002] Bacteria are medically relevant infectious organisms that account for thousands of infections every year, including spore forming bacteria. In particular, bacteria such as Clostridioides difficile (C. difficile), Staphylococcus aureus (S. aureus), and Escherichia coli (E. coli) represent important bacterial species that are capable of infecting animals at a highly virulent rate.

[0003] Typical methods for identification and sequencing of both Gram-positive and Gram-negative bacteria are very laborious. These manual methods, including those based on bead -based techniques, are arduous and can produce limited qualify and / or quantify of isolated nucleic acids. Thus, there exists a need to provide rapid and accurate nucleic acid isolation from bacteria, in particular via automated methods.

[0004] Accordingly, the present disclosure provides novel methods for whole genome sequencing (WGS) of bacteria in order for improving the qualify and quantify of isolated nucleic acids from bacteria. The analyses according to the present disclosure indicate that quality can enhance detection and isolation from bacteria, for instance via automation of WGS. By performing the detailed steps of the present disclosure, a more efficient and higher qualify can be obtained to provide the improved methods described herein.

[0005] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0006] The detailed description particularly refers to the accompanying figures in which:78090-429588-2-

[0007] FIGURE 1 shows the modifications to the methods provided an unexpected increase in nucleic acid concentrations obtained from the bacteria.

[0008] FIGURE 2 shows that the modified protocol yields a higher concentration of DNA for certain bacteria.

[0009] FIGURE 3 shows that the modified protocol yields higher quality DNA for S. aureus compared to the unmodified protocol.

[0010] FIGURE 4 shows that the modified protocol yields an improved A260 / A230 ratio for .S', aureus and for C. difficile compared to the unmodified protocol.

[0011] FIGURE 5 shows that the modified protocol provides quality DNA suitable for WGS to be extracted from S. aureus and for C. difficile compared to the unmodified protocol.

[0012] FIGURE 6 shows the concentration of DNA calculated amongst the various bacteria for unmodified and modified protocols.

[0013] FIGURE 7 shows the A260 / A280 ratio calculated amongst the various bacteria for unmodified and modified protocols.

[0014] FIGURE 8 shows the A260 / A230 ratio calculated amongst the various bacteria for unmodified and modified protocols.

[0015] FIGURE 9 shows the pass / no pass rate per the established criteria amongst the various bacteria for unmodified and modified protocols.DETAILED DESCRIPTION

[0016] Various embodiments of the invention are described herein as follows. In an illustrative aspect, a method of whole genome sequencing of a Gram-positive bacteria is provided. The method comprises the steps of a) isolating nucleic acid from the Gram-positive bacteria to provide a processed sample, and b) preparing a library’ of the isolated nucleic acid from step a).

[0017] In an embodiment, the method is automated. In an embodiment, step a) of the method is automated. In an embodiment, step a) comprises a modified protocol of a QIAamp nucleic acid micro-kit. In an embodiment, step b) of the method is automated. In an embodiment, step b) comprises a modified protocol of an Illumina Nextera nucleic acid prep kit. In an embodiment, step b) comprises use of an automated liquid handling system.

[0018] In an embodiment, the nucleic acid is DNA. In an embodiment, the nucleic acid is RNA.

[0019] In an embodiment, the Gram-positive bacteria is Enterococcus sp. In an embodiment, the Gram-positive bacteria is Enterococcus faecalis. In an embodiment, the78090-429588-3-Gram-positive bacteria is Staphylococcus sp. In an embodiment, the Gram-positive bacteria is Staphylococcus aureus. In an embodiment, the Gram-positive bacteria is Clostridioides difficile.

[0020] In an embodiment, the method comprises a single colony isolation of the Grampositive bacteria prior to step a). In an embodiment, the method comprises culturing the Grampositive bacteria for at least 12 hours immediately prior to step a).

[0021] In an embodiment, the method comprises a pre-reduction of one or more blood agar plates in an anaerobic chamber prior to step a). In an embodiment, the method comprises inoculation of the Gram-positive bacteria to the pre-reduced blood agar plates. In an embodiment, the method comprises incubation of the inoculated pre-reduced blood agar plates in an anaerobic chamber incubator. In an embodiment, the incubation is for at least 6 hours. In an embodiment, the incubation is for at least 8 hours. In an embodiment, the incubation is for at least 12 hours. In an embodiment, the incubation is for at least 16 hours. In an embodiment, the incubation is for at least 20 hours. In an embodiment, the incubation is for less than 24 hours.

[0022] In an embodiment, step a) comprises direct sample collection of the Grampositive bacteria using an inoculating loop. In an embodiment, step a) comprises complete resuspension of Gram-positive bacteria cells in the processed sample. In an embodiment, step a) comprises one or more additional centrifugation steps of the processed sample. In an embodiment, step a) comprises one or more additional incubation steps of the processed sample. In an embodiment, step a) comprises one or more additional vortex steps of the processed sample. In an embodiment, step a) comprises a reduction in Gram-positive bacteria cell density of the processed sample. In an embodiment, step a) comprises an increase in lysis of Gram-positive bacteria cells in the processed sample. In an embodiment, step a) comprises an increase in purity of isolated nucleic acid from the Gram-positive bacteria cells in the processed sample. In an embodiment, step a) comprises an increase in concentration of isolated nucleic acid from the Gram-positive bacteria cells in the processed sample.

[0023] In an embodiment, step b) comprises modification of a tagmentation step. In an embodiment, step b) comprises modification of a library size selection step. In an embodiment, step b) does not comprise use of a Solid-Phase Reversible Immobilization (SPRI) plate.

[0024] In an embodiment, the method further comprises a step of diagnosing an infection in a patient based on identification of the isolated nucleic acid. In an embodiment, the method further comprises a step of treating a patient based on identification of the isolated nucleic acid. In an embodiment, the method further comprises a step of identifying an78090-429588-4- antibiotic resistant gene from the isolated nucleic acid. In an embodiment, the method further comprises a step of analyzing the library of step b) using bioinformatics.

[0025] In an illustrative aspect, a method of whole genome sequencing of a Gramnegative bacteria is provided. The method comprises the steps of a) isolating nucleic acid from the Gram-negative bacteria to provide a processed sample, and b) preparing a library of the isolated nucleic acid from step a).

[0026] In an embodiment, the method is automated. In an embodiment, step a) of the method is automated. In an embodiment, step a) comprises a modified protocol of a QIAamp nucleic acid micro-kit. In an embodiment, step b) of the method is automated. In an embodiment, step b) comprises a modified protocol of an Illumina Nextera nucleic acid prep kit. In an embodiment, step b) comprises use of an automated liquid handling system.

[0027] In an embodiment, the nucleic acid is DNA. In an embodiment, the nucleic acid is RNA.

[0028] In an embodiment, the Gram-negative bacteria is Escherichia sp. In an embodiment, the Gram-negative bacteria is Escherichia coli. In an embodiment, the Gramnegative bacteria is Klebsiella sp. In an embodiment, the Gram-negative bacteria is Acinetobacter sp.

[0029] In an embodiment, the method comprises a single colony isolation of the Gramnegative bacteria prior to step a). In an embodiment, the method comprises culturing the Gramnegative bacteria for at least 12 hours immediately prior to step a). In an embodiment, the method comprises a pre-reduction of one or more blood agar plates in an anaerobic chamber prior to step a).

[0030] In an embodiment, the method comprises inoculation of the Gram-negative bacteria to the pre-reduced blood agar plates. In an embodiment, the method comprises incubation of the inoculated pre-reduced blood agar plates in an anaerobic chamber incubator. In an embodiment, the incubation is for at least 6 hours. In an embodiment, the incubation is for at least 8 hours. In an embodiment, the incubation is for at least 12 hours. In an embodiment, the incubation is for at least 16 hours. In an embodiment, the incubation is for at least 20 hours. In an embodiment, the incubation is for less than 24 hours.

[0031] In an embodiment, step a) comprises direct sample collection of the Gramnegative bacteria using an inoculating loop. In an embodiment, step a) comprises complete resuspension of Gram-negative bacteria cells in the processed sample. In an embodiment, step a) comprises one or more additional centrifugation steps of the processed sample. In an embodiment, step a) comprises one or more additional incubation steps of the processed78090-429588-5- sample. In an embodiment, step a) comprises one or more additional vortex steps of the processed sample. In an embodiment, step a) comprises a reduction in Gram-negative bacteria cell density of the processed sample. In an embodiment, step a) comprises an increase in lysis of Gram-negative bacteria cells in the processed sample. In an embodiment, step a) comprises an increase in purity of isolated nucleic acid from the Gram-negative bacteria cells in the processed sample. In an embodiment, step a) comprises an increase in concentration of isolated nucleic acid from the Gram-negative bacteria cells in the processed sample.

[0032] In an embodiment, step b) comprises modification of a tagmentation step. In an embodiment, step b) comprises modification of a library size selection step. In an embodiment, step b) does not comprise use of a Solid-Phase Reversible Immobilization (SPRI) plate.

[0033] In an embodiment, the method further comprises a step of diagnosing an infection in a patient based on identification of the isolated nucleic acid. In an embodiment, the method further comprises a step of treating a patient based on identification of the isolated nucleic acid. In an embodiment, the method further comprises a step of identifying an antibiotic resistant gene from the isolated nucleic acid. In an embodiment, the method further comprises a step of analyzing the library of step b) using bioinformatics.

[0034] In an illustrative aspect, a method of whole genome sequencing of a spore of a Gram-positive bacteria is provided. The method comprises the steps of a) incubating a stock comprising the spore in an anaerobic chamber to provide a germinated spore, b) isolating nucleic acid from the germinated spore to provide a processed sample, and c) preparing a library of the isolated nucleic acid from step b).

[0035] In an embodiment, the method is automated. In an embodiment, step a) of the method is automated. In an embodiment, step b) of the method is automated. In an embodiment, step c) of the method is automated. In an embodiment, step c) comprises use of an automated liquid handling system.

[0036] In an embodiment, the nucleic acid is DNA. In an embodiment, the nucleic acid is RNA.

[0037] In an embodiment, the Gram-positive bacteria is Clostridioides difficile.

[0038] In an embodiment, the method comprises a single colony isolation of the Grampositive bacteria prior to step a). In an embodiment, the method comprises a pre-reduction of one or more blood agar plates in an anaerobic chamber prior to step b). In an embodiment, the germinated spores are plated on the pre-reduced blood agar plates. In an embodiment, step a) comprises incubation of the inoculated pre-reduced blood agar plates in an anaerobic chamber incubator. In an embodiment, the incubation is for at least 6 hours. In an embodiment, the78090-429588-6- incubation is for at least 8 hours. In an embodiment, the incubation is for at least 12 hours. In an embodiment, the incubation is for at least 16 hours. In an embodiment, the incubation is for at least 20 hours. In an embodiment, the incubation is for less than 24 hours.

[0039] In an embodiment, step b) comprises direct sample collection of the Grampositive bacteria using an inoculating loop. In an embodiment, step b) comprises complete resuspension of Gram-positive bacteria cells in the processed sample. In an embodiment, step b) comprises one or more additional centrifugation steps of the processed sample. In an embodiment, step b) comprises one or more additional incubation steps of the processed sample. In an embodiment, step b) comprises one or more additional vortex steps of the processed sample. In an embodiment, step b) comprises a reduction in Gram-positive bacteria cell density of the processed sample. In an embodiment, step b) comprises an increase in lysis of Gram-positive bacteria cells in the processed sample. In an embodiment, step b) comprises an increase in purity of isolated nucleic acid from the Gram-positive bacteria cells in the processed sample. In an embodiment, step b) comprises an increase in concentration of isolated nucleic acid from the Gram-positive bacteria cells in the processed sample.

[0040] In an embodiment, step c) comprises modification of a tagmentation step. In an embodiment, step c) comprises modification of a library size selection step. In an embodiment, step c) does not comprise use of a Solid-Phase Reversible Immobilization (SPRI) plate.

[0041] In an embodiment, the method further comprises a step of diagnosing an infection in a patient based on identification of the isolated nucleic acid. In an embodiment, the method further comprises a step of treating a patient based on identification of the isolated nucleic acid. In an embodiment, the method further comprises a step of identifying an antibiotic resistant gene from the isolated nucleic acid. In an embodiment, the method further comprises a step of analyzing the library of step b) using bioinformatics.

[0042] In an illustrative aspect, a method of whole genome sequencing of a microorganism is provided. The method comprises the steps of a) isolating nucleic acid from the microorganism to provide a processed sample, and b) preparing a library of the isolated nucleic acid from step a).

[0043] In an embodiment, the method is automated. In an embodiment, step a) of the method is automated. In an embodiment, step a) comprises a modified protocol of a QIAamp nucleic acid micro-kit. In an embodiment, step b) of the method is automated. In an embodiment, step b) comprises a modified protocol of an Illumina Nextera nucleic acid prep kit. In an embodiment, step b) comprises use of an automated liquid handling system.78090-429588-7-

[0044] In an embodiment, the nucleic acid is DNA. In an embodiment, the nucleic acid is RNA.

[0045] In an embodiment, the microorganism is a bacteria. In an embodiment, the bacteria is a Gram-positive bacteria. In an embodiment, the bacteria is a Gram-negative bacteria.

[0046] In an embodiment, the microorganism is a virus. In an embodiment, the virus is a coronavirus. In an embodiment, the microorganism is a fungus.

[0047] In an embodiment, the method comprises a single colony isolation of the microorganism prior to step a). In an embodiment, the method comprises culturing the microorganism for at least 12 hours immediately prior to step a). In an embodiment, the method comprises a pre-reduction of one or more blood agar plates in an anaerobic chamber prior to step a). In an embodiment, the method comprises inoculation of the microorganism to the pre-reduced blood agar plates. In an embodiment, the method comprises incubation of the inoculated pre-reduced blood agar plates in an anaerobic chamber incubator. In an embodiment, the incubation is for at least 6 hours. In an embodiment, the incubation is for at least 8 hours. In an embodiment, the incubation is for at least 12 hours. In an embodiment, the incubation is for at least 16 hours. In an embodiment, the incubation is for at least 20 hours. In an embodiment, the incubation is for less than 24 hours.

[0048] In an embodiment, step a) comprises direct sample collection of the microorganism using an inoculating loop. In an embodiment, step a) comprises complete resuspension of microorganism cells in the processed sample. In an embodiment, step a) comprises one or more additional centrifugation steps of the processed sample. In an embodiment, step a) comprises one or more additional incubation steps of the processed sample. In an embodiment, step a) comprises one or more additional vortex steps of the processed sample. In an embodiment, step a) comprises a reduction in microorganism cell density of the processed sample. In an embodiment, step a) comprises an increase in lysis of microorganism cells in the processed sample. In an embodiment, step a) comprises an increase in purity of isolated nucleic acid from the microorganism cells in the processed sample. In an embodiment, step a) comprises an increase in concentration of isolated nucleic acid from the microorganism cells in the processed sample.

[0049] In an embodiment, step b) comprises modification of a tagmentation step. In an embodiment, step b) comprises modification of a library size selection step. In an embodiment, step b) does not comprise use of a Solid-Phase Reversible Immobilization (SPRI) plate.78090-429588-8-

[0050] In an embodiment, the method further comprises a step of diagnosing an infection in a patient based on identification of the isolated nucleic acid. In an embodiment, the method further comprises a step of treating a patient based on identification of the isolated nucleic acid. In an embodiment, the method further comprises a step of identifying an antibiotic resistant gene from the isolated nucleic acid. In an embodiment, the method further comprises a step of analyzing the library of step b) using bioinformatics.

[0051] The following numbered embodiments are contemplated and are non-limiting:1. A method of whole genome sequencing of a Gram-positive bacteria, the method comprising the steps of a) isolating nucleic acid from the Gram-positive bacteria to provide a processed sample, and b) preparing a library7of the isolated nucleic acid from step a).2. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the method is automated.3. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) of the method is automated.4. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises a modified protocol of a QIAamp nucleic acid micro-kit.5. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step b) of the method is automated.6. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises a modified protocol of an Illumina Nextera nucleic acid prep kit.7. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises use of an automated liquid handling system.8. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the nucleic acid is DNA.9. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the nucleic acid is RNA.10. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the Gram-positive bacteria is Enterococcus sp.11. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the Gram-positive bacteria is Enterococcus faecalis .12. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the Gram-positive bacteria is Staphylococcus sp.78090-429588-9-13. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the Gram-positive bacteria is Staphylococcus aureus.14. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the Gram-positive bacteria is Clostridioides difficile.15. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the method comprises a single colony isolation of the Gram-positive bacteria prior to step a).16. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the method compnses culturing the Gram-positive bacteria for at least 12 hours immediately prior to step a).17. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the method comprises a pre-reduction of one or more blood agar plates in an anaerobic chamber prior to step a).18. The method of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the method comprises inoculation of the Gram-positive bacteria to the pre-reduced blood agar plates.19. The method of clause 18. any other suitable clause, or any combination of suitable clauses, wherein the method comprises incubation of the inoculated pre-reduced blood agar plates in an anaerobic chamber incubator.20. The method of clause 19, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 6 hours.21. The method of clause 19. any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 8 hours.22. The method of clause 19, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 12 hours.23. The method of clause 19. any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 16 hours.24. The method of clause 19, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 20 hours.25. The method of clause 19. any other suitable clause, or any combination of suitable clauses, wherein the incubation is for less than 24 hours.26. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises direct sample collection of the Gram-positive bacteria using an inoculating loop.78090-429588-10-27. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises complete resuspension of Gram-positive bacteria cells in the processed sample.28. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises one or more additional centrifugation steps of the processed sample.29. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises one or more additional incubation steps of the processed sample.30. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises one or more additional vortex steps of the processed sample.31. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises a reduction in Gram-positive bacteria cell density of the processed sample.32. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises an increase in lysis of Gram-positive bacteria cells in the processed sample.33. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises an increase in purity of isolated nucleic acid from the Gram-positive bacteria cells in the processed sample.34. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises an increase in concentration of isolated nucleic acid from the Grampositive bacteria cells in the processed sample.35. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises modification of a tagmentation step.36. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises modification of a library size selection step.37. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein step b) does not comprise use of a Solid-Phase Reversible Immobilization (SPRI) plate.38. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of diagnosing an infection in a patient based on identification of the isolated nucleic acid.39. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of treating a patient based on identification of the isolated nucleic acid.78090-429588-11-40. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of identifying an antibiotic resistant gene from the isolated nucleic acid.41. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of analyzing the library of step b) using bioinformatics.42. A method of whole genome sequencing of a Gram-negative bacteria, the method comprising the steps of a) isolating nucleic acid from the Gram-negative bacteria to provide a processed sample, and b) preparing a library of the isolated nucleic acid from step a).43. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the method is automated.44. The method of clause 42. any other suitable clause, or any combination of suitable clauses, wherein step a) of the method is automated.45. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises a modified protocol of a QIAamp nucleic acid micro-kit.46. The method of clause 42. any other suitable clause, or any combination of suitable clauses, wherein step b) of the method is automated.47. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises a modified protocol of an Illumina Nextera nucleic acid prep kit.48. The method of clause 42. any other suitable clause, or any combination of suitable clauses, wherein step b) comprises use of an automated liquid handling system.49. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the nucleic acid is DNA.50. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the nucleic acid is RNA.51. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the Gram-negative bacteria is Escherichia sp.52. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the Gram-negative bacteria is Klebsiella sp.53. The method of clause 42. any other suitable clause, or any combination of suitable clauses, wherein the Gram-negative bacteria is Acinetobacter sp.78090-429588-12-54. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the method comprises a single colony isolation of the Gram-negative bacteria prior to step a).55. The method of clause 42. any other suitable clause, or any combination of suitable clauses, wherein the method comprises culturing the Gram-negative bacteria for at least 12 hours immediately prior to step a).56. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the method comprises a pre-reduction of one or more blood agar plates in an anaerobic chamber prior to step a).57. The method of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the method comprises inoculation of the Gram-negative bacteria to the pre-reduced blood agar plates.58. The method of clause 57. any other suitable clause, or any combination of suitable clauses, wherein the method comprises incubation of the inoculated pre-reduced blood agar plates in an anaerobic chamber incubator.59. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 6 hours.60. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 8 hours.61. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 12 hours.62. The method of clause 58. any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 16 hours.63. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 20 hours.64. The method of clause 58. any other suitable clause, or any combination of suitable clauses, wherein the incubation is for less than 24 hours.65. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises direct sample collection of the Gram-negative bacteria using an inoculating loop.66. The method of clause 42. any other suitable clause, or any combination of suitable clauses, wherein step a) comprises complete resuspension of Gram-negative bacteria cells in the processed sample.78090-429588-13-67. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises one or more additional centrifugation steps of the processed sample.68. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises one or more additional incubation steps of the processed sample.69. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises one or more additional vortex steps of the processed sample.70. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises a reduction in Gram-negative bacteria cell density of the processed sample.71. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises an increase in lysis of Gram-negative bacteria cells in the processed sample.72. The method of clause 42. any other suitable clause, or any combination of suitable clauses, wherein step a) comprises an increase in purity of isolated nucleic acid from the Gram-negative bacteria cells in the processed sample.73. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises an increase in concentration of isolated nucleic acid from the Gramnegative bacteria cells in the processed sample.74. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises modification of a tagmentation step.75. The method of clause 42. any other suitable clause, or any combination of suitable clauses, wherein step b) comprises modification of a library size selection step.76. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein step b) does not comprise use of a Solid-Phase Reversible Immobilization (SPRI) plate.77. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of diagnosing an infection in a patient based on identification of the isolated nucleic acid.78. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of treating a patient based on identification of the isolated nucleic acid.79. The method of clause 42. any other suitable clause, or any combination of suitable clauses. wherein the method further comprises a step of identifying an antibiotic resistant gene from the isolated nucleic acid.78090-429588-14-80. The method of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of analyzing the library of step b) using bioinformatics.81. A method of whole genome sequencing of a spore of a Gram-positive bacteria, the method comprising the steps of a) incubating a stock comprising the spore in an anaerobic chamber to provide a germinated spore, b) isolating nucleic acid from the germinated spore to provide a processed sample, and c) preparing a library of the isolated nucleic acid from step b).82. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein the method is automated.83. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step a) of the method is automated.84. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step b) of the method is automated.85. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step c) of the method is automated.86. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step c) comprises use of an automated liquid handling system.87. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein the nucleic acid is DNA.88. The method of clause 81. any other suitable clause, or any combination of suitable clauses, wherein the nucleic acid is RNA.89. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein the Gram-positive bacteria is Clostridioides difficile.90. The method of clause 81. any other suitable clause, or any combination of suitable clauses, wherein the method comprises a single colony isolation of the Gram-positive bacteria prior to step a).91. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein the method comprises a pre-reduction of one or more blood agar plates in an anaerobic chamber prior to step b).92. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the germinated spores are plated on the pre-reduced blood agar plates.78090-429588-15-93. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises incubation of the inoculated pre-reduced blood agar plates in an anaerobic chamber incubator.94. The method of clause 93. any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 6 hours.95. The method of clause 93, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 8 hours.96. The method of clause 93. any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 12 hours.97. The method of clause 93, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 16 hours.98. The method of clause 93, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 20 hours.99. The method of clause 93, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for less than 24 hours.100. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises direct sample collection of the Gram-positive bacteria using an inoculating loop.101. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises complete resuspension of Gram-positive bacteria cells in the processed sample.102. The method of clause 81. any other suitable clause, or any combination of suitable clauses, wherein step b) comprises one or more additional centrifugation steps of the processed sample.103. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises one or more additional incubation steps of the processed sample.104. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises one or more additional vortex steps of the processed sample.105. The method of clause 81. any other suitable clause, or any combination of suitable clauses, wherein step b) comprises a reduction in Gram-positive bacteria cell density of the processed sample.78090-429588-16-106. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises an increase in lysis of Gram-positive bacteria cells in the processed sample.107. The method of clause 81. any other suitable clause, or any combination of suitable clauses, wherein step b) comprises an increase in purity of isolated nucleic acid from the Grampositive bacteria cells in the processed sample.108. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises an increase in concentration of isolated nucleic acid from the Gram-positive bacteria cells in the processed sample.109. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein step c) comprises modification of a tagmentation step.110. The method of clause 81. any other suitable clause, or any combination of suitable clauses, wherein step c) comprises modification of a library size selection step.1 11. The method of clause 81 , any other suitable clause, or any combination of suitable clauses, wherein step c) does not comprise use of a Solid-Phase Reversible Immobilization (SPRI) plate.112. The method of clause 81. any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of diagnosing an infection in a patient based on identification of the isolated nucleic acid.113. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of treating a patient based on identification of the isolated nucleic acid.1 14. The method of clause 81, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of identifying an antibiotic resistant gene from the isolated nucleic acid.115. The method of clause 81. any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of analyzing the library of step b) using bioinformatics.116. A method of whole genome sequencing of a microorganism, the method comprising the steps of a) isolating nucleic acid from the microorganism to provide a processed sample, and b) preparing a library of the isolated nucleic acid from step a).117. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the method is automated.78090-429588-17-1 18. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step a) of the method is automated.119. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises a modified protocol of a QIAamp nucleic acid microkit.120. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step b) of the method is automated.121. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises a modified protocol of an Illumina Nextera nucleic acid prep kit.122. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises use of an automated liquid handling system.123. The method of clause 116. any other suitable clause, or any combination of suitable clauses, wherein the nucleic acid is DNA.124. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the nucleic acid is RNA.125. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the microorganism is a bacteria.126. The method of clause 125, any other suitable clause, or any combination of suitable clauses, wherein the bacteria is a Gram-positive bacteria.127. The method of clause 125, any other suitable clause, or any combination of suitable clauses, wherein the bacteria is a Gram-negative bacteria.128. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the microorganism is a virus.129. The method of clause 128, any other suitable clause, or any combination of suitable clauses, wherein the virus is a coronavirus.130. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the microorganism is a fungus.131. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the method comprises a single colony isolation of the microorganism prior to step a).132. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the method comprises culturing the microorganism for at least 12 hours immediately prior to step a).78090-429588-18-133. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the method comprises a pre-reduction of one or more blood agar plates in an anaerobic chamber prior to step a).134. The method of clause 133, any other suitable clause, or any combination of suitable clauses, wherein the method comprises inoculation of the microorganism to the prereduced blood agar plates.135. The method of clause 134, any other suitable clause, or any combination of suitable clauses, wherein the method comprises incubation of the inoculated pre-reduced blood agar plates in an anaerobic chamber incubator.136. The method of clause 135, any other suitable clause. or any combination of suitable clauses, wherein the incubation is for at least 6 hours.137. The method of clause 135, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 8 hours.138. The method of clause 135, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 12 hours.139. The method of clause 135, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 16 hours.140. The method of clause 135, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for at least 20 hours.141. The method of clause 135, any other suitable clause, or any combination of suitable clauses, wherein the incubation is for less than 24 hours.142. The method of clause 116. any other suitable clause, or any combination of suitable clauses, wherein step a) comprises direct sample collection of the microorganism using an inoculating loop.143. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises complete resuspension of microorganism cells in the processed sample.144. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises one or more additional centrifugation steps of the processed sample.145. The method of clause 116. any other suitable clause, or any combination of suitable clauses, wherein step a) comprises one or more additional incubation steps of the processed sample.78090-429588-19-146. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises one or more additional vortex steps of the processed sample.147. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises a reduction in microorganism cell density of the processed sample.148. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises an increase in lysis of microorganism cells in the processed sample.149. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step a) comprises an increase in purity7of isolated nucleic acid from the microorganism cells in the processed sample.150. The method of clause 116. any other suitable clause, or any combination of suitable clauses, wherein step a) comprises an increase in concentration of isolated nucleic acid from the microorganism cells in the processed sample.151. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises modification of a tagmentation step.152. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step b) comprises modification of a library size selection step.153. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein step b) does not comprise use of a Solid-Phase Reversible Immobilization (SPRI) plate.154. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of diagnosing an infection in a patient based on identification of the isolated nucleic acid.155. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of treating a patient based on identification of the isolated nucleic acid.156. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of identifying an antibiotic resistant gene from the isolated nucleic acid.157. The method of clause 116, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises a step of analyzing the library7of step b) using bioinformatics.78090-429588-20-EXAMPLESEXAMPLE 1Modified Isolation of Nucleic Acid from Gram-Positive Bacteria

[0052] Due to the composition of bacterial cell walls, it is difficult to produce nucleic acid (e g., DNA) of a sufficient quality that is suitable for sequencing. The instant example provides exemplary methods for isolation of whole genomic DNA desirable quality and quantity from many different microorganism species.

[0053] Bacterial genome DNA isolation can be performed using a QIAamp DNA Micro-kit (Qiagen) and DNA isolation protocols for Gram-positive bacteria as found in the DNeasy Blood &Tissue kit (Qiagen) handbook. However, for the instant example, a modified method of DNA isolation was performed in order to yield better quality and quantity of DNA isolated for whole genome sequencing.

[0054] One or more of the following numbered modifications can be included in the modified protocol:1. Prior to DNA isolation, single colony isolation can be performed to obtain pure bacterial cells and to remove possible DNA contamination from other microbes that could be found in the primary samples.2. Fresh bacterial cells can be cultured overnight just before DNA isolation.3. Liquid cultures can be replaced with plate cultures. The sample collection procedure can be changed to direct sample collection using an inoculating loop, instead of measuring the OD value and harvesting by centrifugation.4. Cells can be fully resuspended to increase cell lysis efficiency.5. One or more additional wash steps with AW1 can be added, as well as increasing contact time for washing solution AW1 and AW2 to increase the purity of the DNA isolate.6. The incubation time can be increased in the elution step and the volume of elution buffer can be decreased to increase the final DNA concentration.7. For Gram-positive bacteria, reagents volume can be doubled to reduce the cell density to facilitate the resuspension of the cells before cell lysis. With this modification, cell lysis can be improved. Further, the reaction time can be increased for the lysozy me and Proteinase K steps in order to realize complete lysis of the cells.78090-429588-21-8. Centrifuge speed can be increased to facilitate flow-through.

[0055] A comparison of the QIAgen Gram-Positive Bacteria protocol steps and the modified Gram-Positive Bacteria protocol steps is provided in Table 1. The QIAgen protocol is listed in the left column and the modified protocol is listed in the right column, with notes to the numbered modifications provided above.Table 1.78090-429588-22-EXAMPLE 2Modified Isolation of Nucleic Acid from C. difficile

[0056] The protocols provided in Example 1 can be utilized for Clostridioides difficile (C. difficile'). The instant example provides further exemplary modifications for processing C. difficile cells.

[0057] Prior to proceeding with the protocols of Example 1, C. diff cells can be pretreated according to the following numbered modifications.1. Anaerobic blood agar plates can be pre-reduced in an anaerobic chamber for between 6- 24 hours at room temperature.2. C. diff vegetative cells can be inoculated to pre-reduced anaerobic blood agar plates.3. The pre-reduced anaerobic blood agar plates with C. diffcw be incubated in an anaerobic chamber incubator for less than 24 hours (e.g., 18-20 hours).4. Lysozyme buffer (20 mM Tris-HCL, pH 8.0; 2mM EDTA; 1% Triton X-100) can be prepared.5. A thermomixer can be set to 37 °C.6. Approximately 0.01 g of lysozyme powder can be measured on a scale and placed in 15 ml tube.78090-429588-23-7. Approximately 1 ml of Lysozyme buffer (Tris-EDTA and Triton X-100 solution) per 0.01 g of lysozy me powder can be added to the tube.8. The tube can be vortexed to completely dissolve lysozy me powder in the lysozyme buffer (Tris-EDTA and Triton X-100 solution).9. For each sample, a 2 ml screw top tube can be prepared, glass beads (0. 1 mm) can be added, and a 500 pl lysozyme buffer containing lysozyme powder prepared in step 7 can be added.10. Bacteria cells can be added to the tube prepared in step 9.11. The tube can be vortexed vigorously at maximum speed for approximately 5-9 minutes in order to completely re-suspend pellet. The vortex time depends on the number of cells (e.g., check the turbidity ).12. After resuspension, approximately 360 pl of the resuspension can be transferred into a new 1.5 ml microcentrifuge tube.13. The new tube can be incubated at 37 °C for about 1 hour with shaking in a thermomixer.14. After about 1 hour of incubation, the thermomixer temperature can be increased to 56 °C for the following step.15. Approximately 50 pl proteinase K and 400 pl Buffer AL can be added to the tube and vortexed to mix.16. The tube can be incubated at 56 °C for 1 hour with shaking.17. Approximately 400 pl EtOH (96-100%) can be added to the tube. The lid can be closed and pulse-vortexed for about 15 seconds.18. Microcentrifuge tubes can be briefly centrifuged to remove any drops from inside of the lid.19. The entire lysate (sample) can be transferred into a QIAamp MinElute column, without wetting the rim. The lid is closed and the tube can be centrifuged at 12,000 x g for 1 minute. The QIAamp MinElute can be placed in a clean 2 ml collection tube and the collection tube containing the flow-through can be discarded. Repeat steps for remaining lysates.20. About 500 pL of Buffer AW1 can be added to the tube without wetting the rim, letting it sit for about 1 minute. Then the lid can be closed and the tube centrifuged at 12,000 x g for 1 minute. The QIAamp MinElute can be placed in a clean 2 ml collection tube and the collection tube containing the flow-through can be discarded. Repeat this step.21. The QIAamp MinElute column can be opened and 500 pl buffer AW2 can be added without wetting the rim, letting it sit for about 1 minute.78090-429588-24-22. The lid to the tube can be closed lid and the tube can be centrifuged at 12,000 x g for about 1 minute. The QIAamp MinElute can be placed in a clean 2 ml collection tube and the collection tube containing the flow-through can be discarded.23. The tube can be centrifuged at full-speed 12,000 x g for about 3 minutes to dry membrane completely.24. The QIAamp MinElute can be placed in a clean 1.5 ml microcentrifuge tube and the collection tube containing the flow-through can be discarded.25. The lid of the QIAamp MinElute column can be opened and 50 pl of molecular grade distilled water can be added to the center of membrane, letting it sit for about 5 minutes before centrifuging.26. The tube can be centrifuged at full speed 12,000 x g for about 1 minute.27. The item can be read on a nanodrop (e.g., the yield can be greater than 20 ng / pl and 260 / 280 ratio for DNA can be greater than 1.8).

[0058] Furthermore, if the C. diff culture starts from spore stock, then additional modifications to the protocol of the instant example can be made. For instance, approximately 50 pl of spore stock can be plated on CHROMID C. diff plates and then incubated in an anaerobic chamber at 35 °C for at least 48 hours to germinate spores. Thereafter, anaerobic blood agar plates can be pre-reduced in an anaerobic chamber for between 6-24 hours at room temperature. C. diff cells can then be transferred from CHROMID C. diff plates to the prereduced anaerobic agar plates and incubated for about 48 hours in an anaerobic chamber at 35 °C. Finally, the prepared C. diff vegetative cells can be inoculated onto pre-reduced anaerobic agar plates and incubated for about 18-20 hours (e.g., corresponding to steps 1-3 above).EXAMPLE 3Modified Isolation of Nucleic Acid from C. difficile Spores

[0059] Isolation of genomic DNA directly from spores has many advantages, including lack of extensive culture requirement. The breakdown of spore walls is difficult but required in order to obtain high quality DNA for sequencing.

[0060] In this regard, the protocols provided in Example 1 can be utilized for isolation of genomic DNA directly from C. difficile spores. The instant example provides further exemplary modifications for processing C. difficile spores.78090-429588-25-

[0061] The protocols of the instant example achieved comparable sequencing results from spores for direct DNA isolation from C. difficile spores. Table 2 shows the comparison of these results.Table 2.

[0062] As shown in Table 2. the quality of sequencing of the spore-derived DNA was comparable to that of the vegetative cell DNA (a similarity of 99.94%). This indicates that the protocol of the instant example provides isolation of DNA for whole genome sequencing from C. difficile spores.

[0063] Prior to proceeding with the protocols of Example 1, C. diff cells can be pretreated according to the following numbered modifications.1. Lysozyme buffer (20 mM Tris-HCL, pH 8.0; 2mM EDTA; 1% Triton X-100) can be prepared.2. The temperature of the Thermomixer can be set to 37 °C.3. Approximately 0.01 g of lysozyme powder can be measured on a scale and placed in 15 ml tube.4. Approximately 1 ml of lysozyme buffer (Tris-EDTA and Triton X-100 solution) can be added per 0.01 g of lysozy me powder.5. The tube can be vortexed to completely dissolve the lysozyme powder in the lysozyme buffer (Tris-EDTA and Triton X-100 solution).6. The spore sample can be added to approximately 500 pl lysozyme buffer containing lysozyme powder as prepared in step 5.7. The tube can be incubated at 37 °C for about 1-1.5 hours with shaking in Thermomixer.8. A 2 ml screw top tube can be prepare with glass beads then resuspension can be transferred from step 7.78090-429588-26-9. The tube can be vortexed vigorously at maximum speed for approximately 20-30 minutes in Tissue Lyser LT at maximum speed.10. After resuspension, approximately 360 pl of the resuspension can be transferred into a new 1.5 ml microcentrifuge tube.11. After about 1 hour of incubation, the thermomixer temperature can be increased to 56 °C for the following step.12. Approximately 50 pl proteinase K and 400 pl Buffer AL can be added to the tube and vortexed to mix.13. The tube can be incubated at 56 °C for 1 hour with shaking.14. Approximately 400 pl EtOH (96-100%) can be added to the tube. The lid can be closed and pulse- vortexed for about 15 seconds.15. Microcentrifuge tubes can be briefly centrifuged to remove any drops from inside of the lid.16. The entire lysate (sample) can be transferred into a QIAamp MinElute column, without wetting the rim. The lid is closed and the tube can be centrifuged at 12,000 x g for 1 minute. The QIAamp MinElute can be placed in a clean 2 ml collection tube and the collection tube containing the flow-through can be discarded. Repeat steps for remaining lysates.17. About 500 pL of Buffer AW1 can be added to the tube without wetting the rim, letting it sit for about 1 minute. Then the lid can be closed and the tube centrifuged at 12,000 x g for 1 minute. The QIAamp MinElute can be placed in a clean 2 ml collection tube and the collection tube containing the flow-through can be discarded. Repeat this step.18. The QIAamp MinElute column can be opened and 500 pl buffer AW2 can be added without wetting the rim, letting it sit for about 1 minute.19. The lid to the tube can be closed lid and the tube can be centrifuged at 12,000 x g for about 1 minute. The QIAamp MinElute can be placed in a clean 2 ml collection tube and the collection tube containing the flow-through can be discarded.20. The tube can be centrifuged at full-speed 12,000 x g for about 3 minutes to dry membrane completely.21. The QIAamp MinElute can be placed in a clean 1.5 ml microcentrifuge tube and the collection tube containing the flow-through can be discarded.22. The lid of the QIAamp MinElute column can be opened and 50 pl of molecular grade distilled water can be added to the center of membrane, letting it sit for about 5 minutes before centrifuging.78090-429588-27-23. The tube can be centrifuged at full speed 12,000 x g for about 1 minute.24. The item can be read on a nanodrop (e.g., the yield can be greater than 20 ng / pl and 260 / 280 ratio for DNA can be greater than 1.8).EXAMPLE 4Modified Isolation of Nucleic Acid from Gram-Negative Bacteria

[0064] The instant example provides exemplary' methods for isolation of whole genomic DNA desirable quality and quantity from Gram-negative bacteria.

[0065] Bacterial genome DNA isolation can be performed using a QIAamp DNA Micro-kit (Qiagen) and DNA isolation protocols for Gram-positive bacteria as found in the DNeasy Blood &Tissue kit (Qiagen) handbook. However, for the instant example, a modified method of DNA isolation was performed in order to yield better quality' and quantity of DNA isolated for whole genome sequencing.

[0066] One or more of the following numbered modifications can be included in the modified protocol:1. Prior to DNA isolation, single colony isolation can be performed to obtain pure bacterial cells and to remove possible DNA contamination from other microbes that could be found in the primary samples.2. Fresh bacterial cells can be cultured overnight just before DNA isolation.3. Liquid cultures can be replaced with plate cultures. The sample collection procedure can be changed to direct sample collection using an inoculating loop, instead of measuring the OD value and harvesting by centrifugation.4. Cells can be fully resuspended to increase cell lysis efficiency.5. One or more additional wash steps with AW1 can be added, as well as increasing contact time for washing solution AW1 and AW2 to increase the purity of the DNA isolate.6. The incubation time can be increased in the elution step and the volume of elution buffer can be decreased to increase the final DNA concentration.7. For Gram-positive bacteria, reagents volume can be doubled to reduce the cell density to facilitate the resuspension of the cells before cell lysis. With this modification, cell lysis can be improved. Further, the reaction time can be increased for the lysozyme and Proteinase K steps in order to realize complete lysis of the cells.78090-429588-28-8. Centrifuge speed can be increased to facilitate flow-through.

[0067] A comparison of the QIAgen Gram-negative Bacteria protocol steps and the modified Gram- negative Bacteria protocol steps is provided in Table 3. The QIAgen protocol is listed in the left column and the modified protocol is listed in the right column, with notes to the numbered modifications provided above.Table 3.78090-429588-29-EXAMPLE 5Exemplary Automation of Bacterial Nucleic Acid Isolation

[0068] The various protocols can be utilized for automation of bacteria nucleic acids.The instant example provides further exemplary modifications for automation of bacteria DNA as shown in Table 4. A modified protocol for Gram-positive bacteria is found in the left column and a modified protocol for Gram-negative bacteria is found in the right column.Table 4.78090-429588-30-EXAMPLE 6Modified Library Preparation and Pooling for Tagmentation Step

[0069] The instant example provides exemplary methods for library' preparation and pooling, in particular the tagmentation step. Library preparation for sequencing was automated with the Eppendorf epMotion 5075tc. an automated liquid handling system.

[0070] Library preparation and pooling can be performed using the Illumina Nextera DNA prep kit for bacterial WGS with automation. However, for the instant example, a modified method of library' preparation and pooling was performed in order to optimize the yield and efficiency of the library preparation.

[0071] One or more of the following numbered modifications can be included in the modified protocol:1. Vortex speed and time can be reduced in order to minimize contamination risk from splashing.2. Vortex mixing can be replaced with pipetting up and down at STOP tagmentation step in order to provide a more exact tagmentation reaction time.3. The time delay during tagmentation can be reduced in order to increase the efficiency.4. Incubation time can be increased to facilitate magnetic-based clearing during the tagmentation wash step and to reduce the loss during the following wash step.78090-429588-31-5. The wash buffer can be completely removed after each washing in order to increase the washing efficiency.6. The SPRI plate can be removed and the liquid transfer pattern can be adjusted to minimize movement.7. Unnecessary temperature controls can be omitted in order to shorten the reaction time.

[0072] A comparison of the tagmentation protocol steps and the modified tagmentation protocol steps is provided in Table 5. The Illumina DNA Prep protocol is listed in the left column and the modified protocol is listed in the right column, with notes to the numbered modifications provided above. The modification of the tagmentation protocol provides two advantages, namely by optimizing the tagmentation reaction to increase efficiency and also to increase the index PCR efficiency by improving the tagmentation bead washing step after the tagmentation reaction.Table 5.78090-429588-32-78090-429588-33-78090-429588-34-EXAMPLE 7Modified Library Preparation and Pooling for Library Size Selection Step

[0073] The instant example provides exemplary methods for library' preparation and pooling, in particular the library size selection step. Library preparation for sequencing was automated with the Eppendorf epMotion 5075tc, an automated liquid handling system.

[0074] Library' preparation and pooling can be performed using the Illumina Nextera DNA prep kit for bacterial WGS with automation. However, for the instant example, a modified method of library’ preparation and pooling was performed in order to optimize the yield and efficiency of the library preparation.

[0075] One or more of the following numbered modifications can be included in the modified protocol:78090-429588-35-1. Vortex speed and time can be reduced in order to minimize contamination risk from splashing.2. Vortex mixing can be replaced with pipetting up and down at STOP tagmentation step in order to provide a more exact tagmentation reaction time.3. The time delay during tagmentation can be reduced in order to increase the efficiency.4. Incubation time can be increased to facilitate magnetic-based clearing during the tagmentation wash step and to reduce the loss during the following wash step.5. The wash buffer can be completely removed after each washing in order to increase the washing efficiency.6. The SPRI plate can be removed and the liquid transfer pattern can be adjusted to minimize movement.7. Unnecessary temperature controls can be omitted in order to shorten the reaction time.

[0076] A comparison of the library' size selection protocol steps and the modified library size selection protocol steps is provided in Table 6. The Illumina DNA Prep protocol is listed in the left column and the modified protocol is listed in the right column, with notes to the numbered modifications provided above. The modification of the library size selection protocol provides removal of the SPRI plate to reduce maneuvering and consumable consumption.Reducing maneuvering of the automation system can minimize potential for error, in addition to saving time.Table 6.78090-429588-36-78090-429588-37-78090-429588-38-EXAMPLE 8Modified Library Preparation and Pooling for Viral Nucleic Acids

[0077] The instant example provides exemplar}' methods for library preparation and pooling of viral RNA. Library preparation for sequencing was automated with the Eppendorf epMotion 5075tc, an automated liquid handling system.

[0078] Library preparation can be performed using the Illumina COVIDseq kit library preparation protocol with various, separate programs: i. cDNA synthesis, ii. library size selection, and iii. library clean up. However, for the instant example, a modified method was performed for each of the programs in order to optimize the yield and efficiency of the library preparation.

[0079] One or more of the following numbered modifications can be included in the modified protocol:8. The use of mineral oil during cDNA synthesis can be removed to increase the tagmentation efficiency.9. Pipetting height can be adjusted and time delay during tagmentation can be reduced to increase the tagmentation efficiency10. Inaccurate volume, incubation time, and temperature can be corrected.78090-429588-39-11. Vortex speed and time can be reduced in order to reduce contamination risk from splashing.12. Vortex mix can be replaced with pipetting up and down at STOP tagmentation step to provide a more exact tagmentation reaction time.13. A mixing step can be added after indexing primers are added to increase PCR efficiency.14. A manual bead removal step can be added to reduce the contamination of high molecular weights. Accordingly, the library pooling volume can be increased.

[0080] A comparison of the standard protocol steps and the modified protocol steps for cDNA synthesis is provided in Table 7. The Illumina COVIDseq kit protocol is listed in the left column and the modified protocol is listed in the right column, with notes to the numbered modifications provided above. A particular modification is the removal of mineral oil in the annealing step to increase the tagmentation efficiency. The annealing step in this program is performed on thermocycler outside of epMotion 5075tc.Table 7. cDNA synthesis78090-429588-40-78090-429588-41-

[0081] A comparison of the standard protocol steps and the modified protocol steps for tagmentation is provided in Table 8. The Illumina COVIDseq kit protocol is listed in the left column and the modified protocol is listed in the right column, with notes to the numbered modifications provided above. In this step, the amplicons from the previous program are processed to appropriate fragments for sequencing by tagmentation. The following points are achieved by the modifications 1) Tagmentation efficiency is optimized by reducing time delay in between prior and post tagmentation and adequate mixing of stop buffer and 2) Index PCR efficiency is increased by improving tagmentation bead washing steps following the tagmentation reaction.Table 8. Tagmentation78090-429588-42-78090-429588-43-78090-429588-44-

[0082] A comparison of the standard protocol steps and the modified protocol steps for library cleanup is provided in Table 9. The Illumina COVIDseq kit protocol is listed in the left column and the modified protocol is listed in the right column, with notes to the numbered modifications provided above. In this step, libraries can be pooled into a 1.5 ml microcentrifuge tube prior to manual cleanup. To avoid contamination of high molecular weight fragments, an extra manual cleanup step is added, and pooling volume is increased.78090-429588-45-Table 8. Library CleanupEXAMPLE 9Additional Automated WGS Methods

[0083] The instant example provides alternative methods to optimize and automate whole genome sequencing of bacteria. For example, Table 9 shows possible further modifications for automated bacteria DNA isolation from Gram-positive bacteria (e.g., Enterococcus, Staphylococcus') with Qiacube. The standard protocol is listed in the left column and the modified protocol is listed in the right column.Table 9.

[0084] Further, Table 10 shows possible further modifications for automated bacteriaDNA isolation from Clostridiodes difficile with modified protocols of a Monarch (NEB) gDNA78090-429588-46- purification kit. The standard protocol is listed in the left column and the modified protocol is listed in the right column.Table 10.78090-429588-47-EXAMPLE 10Evaluation of Method Steps for WGS of Various Bacteria

[0085] Experiments of whole genome sequencing (WGS) of various bacteria were performed to compare the standard methods of the QIAGEN DNeasy kit to the modified methods described herein. The experiments were performed on E. coll, S. aureus, and C. difficile.

[0086] As shown in Figure 1, the modifications to the methods provided an unexpected increase in nucleic acid concentrations obtained from the bacteria. Of note, the ideal concentration is 20 ng / pl and the ideal ratio for A260 / A230 is 1.8 and above for Gram-negative organisms and 1.3 and above for Gram-positive organisms.EXAMPLE 11Comparison of Unmodified and Modified Protocols - Manual Extraction

[0087] The instant example provides a comparison of manual DNA extraction using i) unmodified (Qiagen DNeasy Bacteria) protocols and ii) modified protocols with QIAmp Micro DNA kit.

[0088] Extracted DNA quantity was measured with two Gram-negative bacteria and three Gram-positive bacteria, including C. difficile. Figure 2 compares the concentrations of DNA extracted via the unmodified (manual) protocol or the modified (automated) protocol of the present disclosure. As shown in Figure 2, the modified protocol yields a higher concentration of DNA for certain bacteria.

[0089] The quality of extracted DNA was assessed with the ratio of A260 / A280 absorbance at Nanodrop. Generally, pure DNA provides a 1.8-2.0 ratio. Figure 3 compares the ratio of A260 / A280 absorbance, indicating the DNA vs. RNA present and an indicator of purity via the unmodified (manual) protocol or the modified (automated) protocol of the present disclosure. As shown in Figure 3, the modified protocol yields higher quality DNA for 5. aureus compared to the unmodified protocol.78090-429588-48-

[0090] The A260 / A230 ratio is secondary measure of assessing DNA purity, for instance as a sensitive indicator of contaminants (e.g., polysaccharides, proteins, solid particles, etc.) that absorb at A230 nm. Figure 4 compares the A260 / A230 ratio via the unmodified (manual) protocol or the modified (automated) protocol of the present disclosure. As shown in Figure 4, the modified protocol yields an improved A260 / A230 ratio for S. aureus and for C. difficile compared to the unmodified protocol.

[0091] Suitable DNA quality that can be used in downstream application such as whole genome sequencing can be established as a quantity > 20 ng / pl, A260 / A280 > 1.8. and / or A260 / A230 > 1.5. The pass rate indicates the percentage DNA samples meet the criteria.

[0092] Figure 5 compares the unmodified (manual) protocol and the modified (automated) protocol of the present disclosure. As shown in Figure 5, the modified protocol provides quality DNA suitable for WGS to be extracted from S. aureus and for C. difficile compared to the unmodified protocol.EXAMPLE 12Comparison of Unmodified and Modified Protocols - Automated Extraction

[0093] The instant example provides a comparison of i) automated DNA extraction with Unmodified (Qiagen DNeasy Bacteria) protocols and ii) modified protocol with QIAmp Micro DNA kit. For C. difficile automation, adaptation of a Monarch (NEB) gDNA purification was used in place of QIAmp Micro DNA kit.

[0094] The DNeasy column that used for unmodified protocol left white pellet was observed in tube after the DNA elution for all Klebsiella manual prep, E. coli and Klebsiella automated prep samples. DNA concentration and quality, especially A260 / A230 ratio is much low er for all these samples with pellet after freeze-thaw. After remeasuring, samples for E. coli and Klebsiella automated prep passed criteria and only 10% of Klebsiella manual prep sample met the criteria.

[0095] No pellet w as observed with modified protocol, indicating that DNA concentration and quality7were maintained. For downstream applications, the quality7of DNA, especially A260 / A230 ratio is important as it indicates contamination of salt that may hamper the sequencing quality.

[0096] Figure 6 shows the concentration of DNA calculated amongst the various bacteria for unmodified and modified protocols. Figure 7 show s the A260 / A280 ratio calculated amongst the various bacteria for unmodified and modified protocols. Figure 8 shows the A260 / A230 ratio calculated amongst the various bacteria for unmodified and modified78090-429588-49- protocols. Figure 9 shows the pass / no pass rate per the established criteria amongst the various bacteria for unmodified and modified protocols.

Claims

78090-429588-50-WHAT IS CLAIMED IS:

1. A method of whole genome sequencing of a Gram-positive bacteria, the method comprising the steps of a) isolating nucleic acid from the Gram-positive bacteria to provide a processed sample, and b) preparing a library of the isolated nucleic acid from step a).

2. The method of claim 1, wherein the method is automated.

3. The method of claim 1. wherein step a) of the method is automated.

4. The method of claim 1, wherein step a) comprises a modified protocol of a QIAamp nucleic acid micro-kit.

5. The method of claim 1, wherein step b) of the method is automated.

6. The method of claim 1 , wherein step b) comprises a modified protocol of an Illumina Nextera nucleic acid prep kit.

7. The method of claim 1, wherein step b) comprises use of an automated liquid handling system.

8. The method of claim 1, wherein the nucleic acid is DNA.

9. The method of claim 1. wherein the nucleic acid is RNA.

10. The method of claim 1, wherein the Gram-positive bacteria isEnterococcus sp.

11. The method of claim 1, wherein the Gram-positive bacteria isStaphylococcus sp.

12. The method of claim 1. wherein the Gram-positive bacteria isClostridioides difficile.

13. The method of claim 1, wherein the Gram-positive bacteria is aClostridioides difficile spore.

14. The method of claim 1, wherein the method comprises a single colony isolation of the Gram-positive bacteria prior to step a).

15. The method of claim 1 , wherein the method comprises culturing the Grampositive bacteria for at least 12 hours immediately prior to step a).

16. The method of claim 1, wherein the method comprises a pre-reduction of one or more blood agar plates in an anaerobic chamber prior to step a).

17. The method of claim 17, wherein the method comprises inoculation of the Gram-positive bacteria to the pre-reduced blood agar plates.78090-429588-51-18. The method of claim 1, wherein step a) comprises direct sample collection of the Gram-positive bacteria using an inoculating loop.

19. The method of claim 1 , wherein step a) comprises complete resuspension of Gram-positive bacteria cells in the processed sample.

20. The method of claim 1, wherein step a) comprises one or more additional centrifugation steps of the processed sample.

21. The method of claim 1. wherein step a) comprises one or more additional incubation steps of the processed sample.

22. The method of claim 1. wherein step a) comprises one or more additional vortex steps of the processed sample.

23. The method of claim 1, wherein step a) comprises a reduction in Grampositive bacteria cell density of the processed sample.

24. The method of claim 1, wherein step a) comprises an increase in lysis of Gram-positive bacteria cells in the processed sample.

25. The method of claim 1, wherein step a) comprises an increase in purity of isolated nucleic acid from the Gram-positive bacteria cells in the processed sample.

26. The method of claim 1, wherein step a) comprises an increase in concentration of isolated nucleic acid from the Gram-positive bacteria cells in the processed sample.

27. The method of claim 1, wherein step b) comprises modification of a tagmentation step.

28. The method of claim 1. wherein step b) comprises modification of a library size selection step.

29. The method of claim 1, wherein step b) does not comprise use of a Solid- Phase Reversible Immobilization (SPRI) plate.

30. The method of claim 1, wherein the method further comprises a step of diagnosing an infection in a patient based on identification of the isolated nucleic acid.

31. The method of claim 1 , wherein the method further comprises a step of treating a patient based on identification of the isolated nucleic acid.

32. The method of claim 1, wherein the method further comprises a step of identifying an antibiotic resistant gene from the isolated nucleic acid.

33. The method of claim 1, wherein the method further comprises a step of analyzing the library' of step b) using bioinformatics.78090-429588-52-34. A method of whole genome sequencing of a Gram-negative bacteria, the method comprising the steps of a) isolating nucleic acid from the Gram-negative bacteria to provide a processed sample, and b) preparing a library of the isolated nucleic acid from step a).

35. The method of claim 34, wherein the method is automated.

36. The method of claim 34, wherein step a) of the method is automated.

37. The method of claim 34, wherein step a) comprises a modified protocol of a QIAamp nucleic acid micro-kit.

38. The method of claim 34, wherein step b) of the method is automated.

39. The method of claim 34, wherein step b) comprises a modified protocol of an Illumina Nextera nucleic acid prep kit.

40. The method of claim 34. wherein step b) comprises use of an automated liquid handling system.

41. The method of claim 34, wherein the nucleic acid is DNA.

42. The method of claim 34, wherein the nucleic acid is RNA.

43. The method of claim 34. wherein the Gram-negative bacteria isEscherichia sp.

44. The method of claim 34, wherein the Gram-negative bacteria is Klebsiella sp.

45. The method of claim 34. wherein the Gram-negative bacteria is Acinetobacter sp.

46. The method of claim 34, wherein the method comprises a single colony isolation of the Gram-negative bacteria prior to step a).

47. The method of claim 34, wherein the method comprises culturing the Gram-negative bacteria for at least 12 hours immediately prior to step a).

48. The method of claim 34, wherein the method comprises a pre-reduction of one or more blood agar plates in an anaerobic chamber prior to step a).

49. The method of claim 48, wherein the method comprises inoculation of the Gram-negative bacteria to the pre-reduced blood agar plates.

50. The method of claim 34, wherein step a) comprises direct sample collection of the Gram-negative bacteria using an inoculating loop.

51. The method of claim 34, wherein step a) comprises complete resuspension of Gram-negative bacteria cells in the processed sample.78090-429588-53-52. The method of claim 34, wherein step a) comprises one or more additional centrifugation steps of the processed sample.

53. The method of claim 34, wherein step a) comprises one or more additional incubation steps of the processed sample.

54. The method of claim 34, wherein step a) comprises one or more additional vortex steps of the processed sample.

55. The method of claim 34, wherein step a) comprises a reduction in Gramnegative bacteria cell density of the processed sample.

56. The method of claim 34. wherein step a) comprises an increase in lysis of Gram-negative bacteria cells in the processed sample.

57. The method of claim 34, wherein step a) comprises an increase in purity' of isolated nucleic acid from the Gram-negative bacteria cells in the processed sample.

58. The method of claim 34, wherein step a) comprises an increase in concentration of isolated nucleic acid from the Gram-negative bacteria cells in the processed sample.

59. The method of claim 34, wherein step b) comprises modification of a tagmentation step.

60. The method of claim 34, wherein step b) comprises modification of a library size selection step.

61. The method of claim 34, wherein step b) does not comprise use of a Solid- Phase Reversible Immobilization (SPRI) plate.

62. The method of claim 34. wherein the method further comprises a step of diagnosing an infection in a patient based on identification of the isolated nucleic acid.

63. The method of claim 34, wherein the method further comprises a step of treating a patient based on identification of the isolated nucleic acid.

64. The method of claim 34. wherein the method further comprises a step of identifying an antibiotic resistant gene from the isolated nucleic acid.

65. The method of claim 34, wherein the method further comprises a step of analyzing the library of step b) using bioinformatics.

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