Compositions and methods for enriching populations of nucleic acids
This disclosure provides methods and compositions that are useful for enriching a particular population of nucleic acids (a “population of interest”) within a complex mixture of nucleic acids. The population of interest may make up a minor portion of a complex mixture of nucleic acids. The methods and compositions provided herein are useful for detecting, predicting, diagnosing, or monitoring a disease or disorder, particularly a disease or disorder caused by a foreign microbe or pathogen.
1. A method of enriching microbial cell-free nucleic acids in a plasma sample obtained from a mammalian host, the method comprising:
(a) providing the plasma sample from the mammalian host, wherein the plasma sample comprises cell-free nucleic acids comprising mammalian host cell-free nucleic acids and the microbial cell-free nucleic acids; and
(b) removing or physically isolating cell-free nucleic acids of one or more length ranges from the plasma sample, thereby enriching the plasma sample for microbial cell-free nucleic acids in the length interval ranges of:
(i) from about 10 to about 60 base pairs,
(ii) from about 240 to about 280 base pairs,
(iii) from about 425 to about 475 base pairs, or
(iv) any combination thereof.
2. The method of claim 1 , wherein the cell-free nucleic acids comprise cell-free DNA; and wherein (b) comprises removing or physically isolating cell-free DNA of one or more length ranges from the plasma sample, thereby enriching the plasma sample for microbial cell-free DNA in the length interval ranges of (i) from about 10 to about 60 base pairs; (ii) from about 240 to about 280 base pairs; or (iii) from about 425 to about 475 base pairs, or (iv) any combination thereof.
3. The method of claim 1 , wherein (b) comprises removing or physically isolating cell-free nucleic acids of length intervals of about 180 base pairs or multiples thereof or of about 360 base pairs or multiples thereof.
4. The method of claim 1 , wherein (b) comprises removing or physically isolating cell-free nucleic acids of length intervals selected from the group consisting of 150 base pairs or multiples thereof, 160 base pairs or multiples thereof, 170 base pairs or multiples thereof, 190 base pairs or multiples thereof, and any combination thereof.
5. The method of claim 1 , wherein the plasma sample comprises at least five microbial cell-free nucleic acid sequences and the method further comprises detecting the at least five microbial cell-free nucleic acid sequences.
6. The method of claim 1 , wherein the mammalian host is a human host.
7. The method of claim 1 , wherein the microbial cell-free nucleic acids are derived from one or more bacterial, viral, fungal, or parasitic genomes.
8. The method of claim 1 , wherein the microbial cell-free nucleic acids are derived from bacteria.
9. The method of claim 1 , wherein the microbial cell-free nucleic acids are derived from viruses.
10. The method of claim 1 , wherein the microbial cell-free nucleic acids are derived from at least one bacterium selected from the group consisting of Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Staphylococcus aureus, Streptococcus, Treponema, Vibrio , and Yersinia.
11. The method of claim 1 , wherein the microbial cell-free nucleic acids are derived from at least one fungus selected from the group consisting of Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis , and Stachybotrys.
12. The method of claim 1 , wherein the microbial cell-free nucleic acids are derived from at least one drug-resistant microbial strain selected from the group consisting of Clostridium difficile , carbapenem-resistant Enterobacteriaceae (CRE), drug-resistant Neisseria gonorrhoeae (cephalosporin resistant), multidrug-resistant Acinetobacter , drug-resistant Campylobacter , fluconazole-resistant Candida , extended spectrum B-lactamase producing Enterobacteriaceae (ESBLs), Vancomycin-resistant Enterococcus (VRE), multidrug-resistant Pseudomonas aeruginosa , drug-resistant non-typhoidal Salmonella , drug-resistant Salmonella Typhi , drug-resistant Shigella , methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant Streptococcus pneumonia , drug-resistant tuberculosis (multidrug-resistant (MDR) and extensively drug-resistant (XDR)), multi-drug resistant Staphylococcus aureus , Vancomycin resistant Staphylococcus aureus (VRSA), erythromycin-resistant Streptococcus Group A, and clindamycin-resistant Streptococcus Group B.
13. The method of claim 1 , wherein the microbial cell-free nucleic acids are derived from at least one virus selected from the group consisting of Adenovirus, Amur virus, Andes virus, Animal virus, Astrovirus, Avian nephritis virus, Avian Orthoreovirus, Avian Reovirus, Banna virus, Bas-Congo virus, Bat-borne virus, BK virus, Blueberry shock virus, Chicken anaemia virus, Bovine adenovirus, Bovine coronavirus, Bovine herpesvirus 4, Bovine parvovirus, Bulbul coronavirus HKU11, Carrizal virus, Catacamas virus, Chandipura virus, Channel catfish virus, Choclo virus, Coltivirus, Cricket paralysis virus, Crimean-Congo hemorrhagic fever virus, Dobrava-Belgrade virus, El Moro Canyon virus, Elephant endotheliotropic herpesvirus, Epstein-Barr virus, Feline leukemia virus, Foot-and-mouth disease virus, Gou virus, Guanarito virus, Hantavirus, human coronavirus EMC/2012 (HCoV-EMC/2012), Hendra virus, Henipa virus, Hepatitis D, Hepatitis E virus, Human bocavirus, Human herpesvirus type 8, Human metapneumovirus, Imjin virus, Isla Vista virus, JC virus, Junin virus, Khabarovsk virus, Koi herpes virus, Kuniin virus, Lassa virus, Limestone Canyon virus, Lloviu cuevavirus, Lloviu virus, Lujo virus, Machupo virus, Magboi virus, Marburg virus, Melaka virus, Menangle virus, Middle East respiratory syndrome coronavirus, Miniopterus Bat coronavirus 1, Miniopterus Bat coronavirus HKU8, Monkeypox virus, Monongahela virus, Muju virus, Nipah virus, Norovirus group 1, Norovirus group II, Orbivirus, Phytoreovirus, Pipistrellus bat coronavirus HKU5, Porcine adenovirus, Prospect Hill virus, Qalyub virus, Ravn virus, Reston virus, Reticuloendotheliosis virus, Rhinolophus Bat coronavirus HKU2, Roseolovirus, Ross River virus, Rotavirus, Rousettus bat coronavirus HKU9, Saaremaa virus, Sabia virus, Sangassou virus, Sapovirus, Scotophilus Bat coronavirus 512, Serang virus, Severe acute respiratory syndrome virus, Shope papilloma virus, Simian foamy virus, Sin Nombre virus, Smallpox virus, Soochong virus, Sudan ebolavirus, Sudan virus, Tai Forest ebolavirus, Tai Forest virus, Tanganya virus, Thottapalayam virus, Topografov virus, Tremovirus, Tula virus, Turkey coronavirus, Turkeypox virus, Tvlonycteris bat coronavirus HKU4, West Nile virus, Woodchuck hepatitis virus, Zika virus, and Zaire ebolavirus.
14. The method of claim 1 , wherein the microbial cell-free nucleic acids are derived from a pathogen selected from the group consisting of a virus, a bacterium, a fungus, a parasite, and a protozoa.
15. The method of claim 1 , wherein the microbial cell-free nucleic acids are derived from at least one pathogen selected from the group consisting of Acanthamoeba, Acari, Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae, Propionibacterium propionicus, Actinomycetona, Eumycetona , Adenoviridae, Alphavirus, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Necator americanus, Angiostrongylus costaricensis, Anisakis , Arachnida Ixodidae, Argasidae, Arcanobacterium haemolyticum, Archiacanthocephala, Moniliformis moniliformis , Arenaviridae, Ascaris lumbricoides, Aspergillus genus, Babesia B. divergens, B. bigemina, B. equi, B. microfti, B. duncani, Babesia genus, Bacillus cereus, Bacteroides genus, Balamuthia mandrillaris, Balantidium coli, Bartonella hemselae, Baylisascaris genus, Baylisascaris procyonis, Bertiella mucronata, Bertiella studeri , BK virus, Blastocystis, Blastocystis hominis, Bordetella pertussis, Brugia malayi, Brugia timori , Bunyaviridae, Burkholderia cepacia, Burkholderia species, Caliciviridae, Cestoda, Taenia multiceps, Chlamydophila pneumoniae, Chlamydophila psittaci , Cimicidae Cimex lectularius, Clonorchis sinensis, Clonorchis viverrini, Clostridium difficile, Clostridium perfiringens, Coccidioides posadasii, Cochliomyia hominivorax , Colorado tick fever virus (CTFV), Coronaviridae, Corynebacterium diphtheriae, Coxiella burnetii , Crimean-Congo hemorrhagic fever virus, Cryptosporidium, Cyclospora cayetanensis, Demodex folliculorum/brevis/canis , Flaviviruses, Dermatobia hominis, Dicrocoelium dendriticum, Dientamoeba fragilis, Dioctophyme renale, Diphyllobothrium, Diphyllobothrium latum, Dracunculus medinensis, Echinococcus genus, Echinococcus granulosus, Echinococcus multilocularis, E. vogeli, E. Oligarthrus, Ehrlichia chafeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterobius vermicularis, Enterobius gregorii , Coxsackie A virus, Enterovirus 71 (EV71), Epidermophyton floccosum, Trichophyton rubrum, Trichophyton mentagrophytes , Epstein-Barr Virus (EBV), Escherichia coli 0157: H7, 0111 and 0104: H4, Fasciola gigantica, Fasciolopsis buski , Filarioidea superfamily, Filoviridae, Flaviviridae, Fonsecaea pedrosoi, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Gnathostoma spinigerum, Gnathostoma hispidum , Group A Streptococcus , Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Halicephalobus gingivalis , Heartland virus, Hepadmaviridae, Hepatitis D Virus, Hepatitis E Virus, Hepeviridae, Hortaea werneckii , Human bocavirus (HBOV), Human herpesvirus 6 (HHV-6), Human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Hymenolepis nana, Hymenolepis diminuta, Isospora belli , JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Lassa virus, Linguatula serrata, Loa loafilaria, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia genus, Mansonella streptocerca, Marburg virus, Metagonimus yokagawai , Microsporidia phylum, Middle East respiratory syndrome coronavirus, Molluscum contagiosum virus (MCV), Monkeypox virus, Mucorales order (Mucormycosis), Entomophthorales order (Entomophthoramycosis), Mumps virus, Mycobacterium lepromatosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Nocardia asteroides, Nocardia species, Oestroidea, Caliphoridae, Sarcophagidae, Onchocerca volvulus, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis Sinensis, Paracoccidioides brasiliensis, Paragonimus africanus, Paragonimus caliensis, Paragonimus kellicotti, Paragonimus skriabini, Paragonimus uterobilateralis, Paragonimus westermani, Paragonimus species, Paramyxoviridae, parasitic dipterous fly larvae, Parvovirus B 19, Pasteurella genus, Pediculus humanus, Pediculus humanus capitis, Pediculus humanus corporis, Phthirus pubis , Piedraiahortae, Plasmodium vivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium knowlesi, Pneumocystis jirovecii , Polyomaviridae, Poxviridae, Prevotella genus, PRNP Pthirus pubis, Pulex irritans , Reoviridae, Rhabdoviridae, Rhinosporidium seeberi , coronaviruses, Rift Valley fever virus, Sabia, Salmonella enterica Subsp. enterica , serovar typhi, Salmonella genus, Sarcocystis bovihominis, Sarcocystis suihominis, Sarcoptes scabiei , SARS coronavirus, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni and Schistosoma intercalatum, Schistosoma mekongi, Shigella genus, Sin Nombre virus, Spirometra erinaceieuropaei, Sporothrix schenckii, Strongyloides stercoralis, Taenia genus, Taenia saginata, Taenia solium , the bacterial family Enterobacteriaceae, Thelazia californiensis, Thelazia callipaeda , Togaviridae, Toxocara canis, Toxocara cati, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, Trichobilharzia regenti , Schistosomatidae, Trichophyton genus, Trichophyton tonsurans, Trichosporon beigelii, Trichuris trichiura, Trichuris trichiura, Trichuris vulpis, Trypanosoma brucei, Tunga penetrans , Urea plasma urealyticum, Variola major, Variola minor , Venezuelan equine encephalitis virus, Vibrio cholerae , West Nile virus, Wuchereria bancrofti, Wuchereria bancrofti, Yersinia enterocolitica, Yersinia pestis , and Yersinia pseudo tuberculosis.
16. The method of claim 1 , further comprising analyzing the microbial cell-free DNA and identifying infectious or pathogenic microbes in the mammalian host based on the microbial cell-free DNA.
17. The method of claim 1 , wherein the microbial cell-free nucleic acids are derived from at least one microbe selected from the group consisting of: Bordetella, Escherichia, Francisella, Pseudomonas, Salmonella, Shigella, Vibrio, Yersinia, Aspergillus, Pneumocystis, Stachybotrys , Anellovirus, and Bunyaviridae.
18. The method of claim 17 , wherein the microbial cell-free nucleic acids comprise cell-free DNA.
19. The method of claim 17 , wherein the at least one microbe is at least one bacterium selected from the group consisting of Bordetella, Escherichia, Francisella, Pseudomonas, Salmonella, Shigella, Vibrio , and Yersinia.
20. The method of claim 17 , wherein the at least one microbe is at least one fungus selected from the group consisting of: Aspergillus, Pneumocystis , and Stachybotrys.
21. The method of claim 17 , wherein the at least one microbe is at least one virus selected from the group consisting of: Anellovirus and Bunyaviridae.
22. The method of claim 1 , further comprising analyzing the microbial cell-free nucleic acids and identifying infectious or pathogenic microbes in the mammalian host based on the microbial cell-free nucleic acids.
23. The method of claim 20 , wherein the analyzing comprises performing next generation sequencing of the microbial cell-free nucleic acids, to generate microbial cell-free nucleic acid sequence reads.
24. The method of claim 1 , wherein the cell-free nucleic acids do not comprise an artificially fragmented nucleic acid.
25. The method of claim 1 , comprising enriching the plasma sample for microbial cell-free nucleic acids in the length interval range of from about 10 to about 60 base pairs.
26. The method of claim 1 , comprising enriching the plasma sample for microbial cell-free nucleic acids in the length interval range of from about 240 to about 280 base pairs.
27. The method of claim 1 , comprising enriching the plasma sample for microbial cell-free nucleic acids in the length interval range of from about 425 to about 475 base pairs.
28. The method of claim 1 , wherein the removing or physically isolating in (b) comprises performing electrophoresis.
29. The method of claim 1 , wherein the removing or physically isolating in (b) comprises binding the cell-free nucleic acids to a solid support.
30. The method of claim 1 , wherein the solid support comprises magnetic beads.