Identifying presence and composition of cell-free nucleic acids

Abstract

This disclosure describes example techniques and systems for identifying the presence and/or composition of nucleic acids in the blood of a host organism of a model species harboring tissue of a donor organism of another species. For example, the technique may involve identifying the presence and composition of nucleic acids in the blood of a mouse harboring tissue of a human or another companion animal. These cell-free nucleic acids that are identified can be used as biomarkers to determine the presence of a disease, its biological behavior, its rate of progression, and/or the response of the disease to one or more unique therapies. In other examples, the cell-free nucleic acids may be used as biomarkers to determine a response of the host species to the tissue of the donor organism or a response of tissue derived from the second organism to transplantation within the first organism of the first species.

What is claimed is: 
     
         1 . A method comprising:
 xenografting tissue from a donor organism of a second species on to a host organism of a first species;   obtaining a sample derived from the host organism, wherein the sample comprises a plurality of molecules of messenger ribonucleic acid (mRNA);   determining, for substantially each molecule of the plurality of molecules of mRNA, a corresponding RNA sequence;   generating a combined dataset of RNA sequence reads by aligning each RNA sequence to a combined reference genome, wherein aligning each RNA sequence to the combined reference genome includes comparing a genomic location of each corresponding RNA sequence with a genomic location of a gene sequence of the combined reference genome, wherein the combined reference genome includes one or more gene sequences from at least a portion of a first genome derived from the first species and at least a portion of a second genome derived from the second species, and wherein the combined dataset of RNA sequence reads includes RNA sequence reads from both the first species and the second species;   filtering non-unique RNA sequence reads from the combined dataset by identifying species-specific RNA sequences exclusive to either the first species or the second species, wherein identifying species-specific RNA sequences includes determining, for each corresponding RNA sequence, whether the RNA sequence is substantially aligned with exactly one corresponding gene sequence of the combined reference genome;   at least one of:
 differentiating an origin species of each species-specific RNA sequence in the filtered combined dataset by determining whether the corresponding RNA sequence is aligned to a gene sequence of the combined reference genome associated with the first genome of the first species or the second genome of the second species, or 
 quantifying an abundance level of each species-specific RNA sequence in the sample by determining an approximate number of times that each RNA sequence substantially aligned with exactly one corresponding gene occurs in the sample; and 
   determining, based on one or more of the differentiation of the origin species or the quantification of the abundance level, that the tissue derived from the donor organism contains a biomarker indicative of at least one of:
 a disease status, 
 a response of the host organism to the tissue derived from the donor organism, 
 a response of tissue derived from the donor organism to transplantation within the host organism, or 
 a response of the host organism to therapy administered to the host organism. 
   
     
     
         2 . The method of  claim 1 , wherein the one or more gene sequences comprise at least one of one or more coding sequences or one or more regulatory sequences. 
     
     
         3 . The method of  claim 1 , further comprising generating the combined reference genome. 
     
     
         4 . The method of  claim 3 , wherein generating the combined reference genome comprises:
 identifying, for each of the one or more gene sequences, a corresponding location within the combined reference genome; and   annotating, for each of the one or more gene sequences, the corresponding location indicates the origin species of the corresponding gene sequence.   
     
     
         5 . The method of  claim 1 , further comprising, for each species-specific RNA sequence, determining that the exactly one corresponding gene sequence is associated with a predetermined cluster of gene sequences. 
     
     
         6 . The method of  claim 5 , wherein the predetermined cluster of gene sequences comprises a group of genes sharing one or more functional characteristics. 
     
     
         7 . The method of  claim 6 , wherein the one or more functional characteristics comprises one or more biological processes or canonical pathways. 
     
     
         8 . The method of  claim 7 , wherein the one or more biological processes or functional characteristics comprise one or more of transcriptional regulation, intracellular signaling, intercellular signaling, cell apoptosis, biomolecule metabolism, biomolecule synthesis, RNA processing, or macromolecule assembly. 
     
     
         9 . The method of  claim 1 ,
 wherein the donor organism contains a biomarker indicative of the disease status, and   wherein the biomarker comprises a nucleic acid sequence associated with a disease.   
     
     
         10 . The method of  claim 1 ,
 wherein the donor organism contains a biomarker indicative of the disease status, and   wherein the disease status comprises at least one of:
 the presence or absence of a disease state, one or more characteristics of an existing disease state, 
 a likelihood of a future progression of an existing disease state, or 
 one or more characteristics of a predicted future progression of an existing disease state. 
   
     
     
         11 . The method of  claim 1 , further comprising determining, based on determining that the tissue derived from the donor organism contains the biomarker indicative of the disease status, a therapy to be administered to at least one of the host organism or the donor organism. 
     
     
         12 . The method of  claim 11 , further comprising administering the determined therapy to the at least one of the host organism or the donor organism. 
     
     
         13 . The method of  claim 1 ,
 wherein the donor organism contains a biomarker indicative of a response to the tissue derived from the donor organism, and   wherein the response of the host organism to the tissue derived from the donor organism corresponds to one of acceptance or rejection of the tissue derived from the donor organism by the host organism.   
     
     
         14 . The method of  claim 1 , wherein obtaining the sample of bodily fluid derived from the host organism comprises:
 obtaining a sample of blood;   isolating, from the sample of blood, a volume of blood serum; and   isolating, from the volume of blood serum, a plurality of exosomes.   
     
     
         15 . The method of  claim 1 , further comprising:
 isolating, from the sample of bodily fluid, the plurality of molecules of mRNA;   purifying the molecules of mRNA;   performing a reverse-transcriptase polymerase chain reaction using the molecules of RNA to produce a plurality of molecules of complementary deoxyribonucleic acid (cDNA), wherein each molecule of the plurality of molecules of cDNA corresponds to one of the plurality of molecules of mRNA;   performing a polymerase chain reaction to amplify the molecules of cDNA;   transcribing substantially each of the molecules of cDNA into RNA; and   determining the nucleic acid sequence of substantially each of the molecules of mRNA.   
     
     
         16 . The method of  claim 1 ,
 wherein the first species comprises one of a rodent species or a non-human primate species, and   wherein the second species comprises one of a canine, feline, porcine, or human species.   
     
     
         17 . A method comprising:
 xenografting tissue from a donor organism of a second species on to a host organism of a first species;   obtaining a sample derived from the host organism wherein the sample comprises a plurality of molecules of messenger ribonucleic acid (mRNA);   generating a combined reference genome, wherein the combined reference genome comprises one or more gene sequences from: at least a portion of a first genome derived from the first species and at least a portion of the second genome derived from the second species;   determining, for substantially each molecule of the plurality of molecules of mRNA, a corresponding RNA sequence;   generating a combined dataset of RNA sequence reads by aligning each RNA sequence to the combined reference genome, wherein aligning each RNA sequence to the combined reference genome includes comparing a genomic location of each corresponding RNA sequence with a genomic location of a gene sequence of the combined reference genome, and wherein the combined dataset of RNA sequence reads includes RNA sequence reads from both the first species and the second species;   filtering non-unique RNA sequence reads from the combined dataset by identifying species-specific RNA sequences exclusive to either the first species or the second species, wherein identifying species-specific RNA sequences includes determining, for each corresponding RNA sequence, whether the RNA sequence is substantially aligned with exactly one corresponding gene sequence of the combined reference genome.   
     
     
         18 . The method of  claim 17 , wherein generating the combined reference genome further comprises:
 identifying, for each of the one or more gene sequences of the combined reference genome, a corresponding location within the combined reference genome; and   annotating, for each of the one or more gene sequences of the combined reference genome, the corresponding location to indicate the origin species of the corresponding gene sequence.   
     
     
         19 . The method of  claim 17 , wherein generating the combined reference genome further comprises:
 receiving data indicating gene sequences of at least a portion of the first genome derived from the first species;   receiving data indicating gene sequences of at least a portion of the second genome derived from the second species; and   outputting one or more computer files representing the one or more gene sequences of the combined reference genome.   
     
     
         20 . The method of  claim 17 , further comprising at least one of:
 differentiating an origin species of each species-specific RNA sequence in the filtered combined dataset by determining whether the corresponding RNA sequence is aligned to a gene sequence of the combined reference genome associated with the first genome of the first species or the second genome of the second species, or   quantifying an abundance level of each species-specific RNA sequence in the sample by determining an approximate number of times that each RNA sequence substantially aligned with exactly one corresponding gene occurs in the sample.