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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
  • G16B50/30—Data warehousing; Computing architectures
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
  • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
  • G16C20/60—In silico combinatorial chemistry
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
  • G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR

Definitions

• the invention relates to methods for identifying T-cell receptor antibody in a population of cells and methods for using that information to measure immune status of a patient and predict the likelihood of which disease the patient might have.
• the human genome comprises a total number of 567-588 IG
• IG and 228-234 TR per haploid genome, localized in the 7 major loci. They comprise 405-418 V, 32 D, 105- 109 J and 25-29 C genes. The number of functional IG and TR genes is 321-353 per haploid genome. They comprise 187-216 V, 28 D, 86-88 J and 20-21 C genes (http://imgt.cines.fr). Through rearrangement of these genes, an estimated 2.5x10 7 possible antibodies or T cell receptors can be generated.
• the invention relates to a method for evaluating changes in immune response cell populations and associating those changes with a specific disease.
• the method comprises the steps of (a) isolating a
• the subpopulation may comprise a whole blood population or another mixed population sample.
• the step of isolating a subpopulation of white blood cells may be performed by flow cytometry to separate naive B cells, mature B cells, memory B cells, naive T cells, mature T cells, and memory T cells.
• the recombinations in the subpopulation of cells are rearrangements of B-cell immunoglobulin heavy chain (IgH), kappa and/or lambda light chains (IgK, IgL), T-cell receptor Alpha, Beta, Gamma, Delta.
• IgH B-cell immunoglobulin heavy chain
• IgK kappa and/or lambda light chains
• T-cell receptor Alpha Beta
• Gamma Delta
• the method may optionally comprise an additional step comprising (g) comparing the rearrangements identified for a population of individuals to whom a vaccine has been administered with the rearrangements identified for a population of individuals to whom the vaccine was not administered to evaluate the efficacy of the vaccine in producing an immune response.
• the method may also optionally comprise the additional step of (g) comparing the rearrangements identified for a population of normal individuals with the rearrangements identified for a population of individuals who have been diagnosed with a disease to determine if there is a correlation between a specific rearrangement or set of rearrangements and the disease.
• the method can produce semi-quantitative amplification of polynucleotides comprising complementarity determining region 3 (CDR3s), which result from genetic rearrangements within T or B cells and are responsible for the affinity and specificity of antibodies and/or T cell receptors for specific antigens.
• CDR3s complementarity determining region 3
• Semi-quantitative amplification provides a method to not only detect the presence of specific CDR3 sequences, but also determine the relative abundance of cells which have produced the necessary recombination events to produce those CDR3 sequences.
• One aspect of the invention therefore relates to a method for analyzing semiquantitative sequence information to provide one or more immune status reports for a human or animal.
• the method for producing an immune status report comprising the steps of (a) identifying one or more distinct CDR3 sequences that are shared between a subject's immunoprofile and a cumulative immunoprofile from a disease library stored in a database, summing a total number of a subject's detected sequences corresponding to those shared distinct CDR3 sequences, and computing the percentage of the total number of detected sequences in the subject's
• Figure 1 a and Figure 1 b are photographs of a gel illustrating the presence of amplification products obtained by the method of the invention using primers disclosed herein.
• Figure 2a and Figure 2b are cartoons representing the observed difference in diversity between an immunoprofile in an individual with a disease and an individual who is generally healthy, with each filled circle representing a distinct CDR3 sequence and the size of the circle representing the number of times that the distinct CDR3 sequence is found in the immunoprofile.
• Figure 3 is a diagram illustrating the method for generating a public library.
• Figure 4 is a diagram illustrating the method for generating a disease library.
• Figure 5 illustrates results obtained by comparing a patient immunoprofile with a disease library, calculating a percentage for each distinct CDR3 in the patient immunoprofile that is shared between the two, and adding those percentages to produce a sum, or sharing index.
• Figure 6 illustrates results obtained by comparing a patient immunoprofile with a subset of a public library, calculating a percentage for each distinct CDR3 that is shared between the two, and adding those percentages in the patient
• Figure 7 is a graph illustrating the method of the invention, where the area under the curve represents total sharing indices obtained for subsets of a public library (sub-libraries), a P-value is estimated, and sharing indices for comparisons of an individual's immunoprofile and one or more disease libraries are represented by vertical lines (DL-i , DL 2 , etc.).
• the inventors have developed methods for evaluating antibody and T cell receptor rearrangements from a large number of cells, the methods being useful for comparing rearrangements identified in populations of individuals to determine whether there is a correlation between a specific rearrangement or set of rearrangements and a disease, or certain symptoms of a disease.
• the method is also useful for establishing a history of the immune response of an individual or individuals in response to infectious and/or environmental agents, as well as for evaluating the efficacy of vaccines.
• the invention relates to a method for evaluating changes in immune response cell populations and associating those changes with a specific disease.
• the method comprises the steps of (a) isolating a
• the subpopulation may comprise a whole blood population or another mixed population sample.
• a peripheral blood sample is taken from a patient and the step of isolating a subpopulation of white blood cells may be performed by flow cytometry to separate naive B cells, mature B cells, memory B cells, naive T cells, mature T cells, and memory T cells.
• the recombinations in the subpopulation of cells are rearrangements of B-cell immunoglobulin heavy chain (IgH), kappa and/or lambda light chains (IgK, IgL), T-cell receptor Beta, Gamma, or Delta.
• the method may comprise an additional step (g) comparing the rearrangements identified for a population of normal individuals with the rearrangements identified for a population of individuals who have been diagnosed with a disease to determine if there is a correlation between a specific rearrangement or set of rearrangements and the disease.
• the method may comprise an additional step comprising (g) comparing the rearrangements identified for a population of individuals to whom a vaccine has been administered with the rearrangements identified for a population of individuals to whom the vaccine was not administered to evaluate the efficacy of the vaccine in producing an immune response.
• the step of separating the amplicons from the first amplification reaction from one or more unused primers from the first amplification reaction may be omitted and the two amplification reactions may be performed in the same reaction tube.
• an apparatus for detecting target polynucleotides in a sample comprising a first amplification chamber for thermocycling to amplify one or more target polynucleotides to produce amplicons using nested primers, at least a portion of the nested primers comprising additional nucleotides to incorporate into a resulting amplicon a binding site for a communal primer; a means for separating the amplicons from the first amplification reaction from one or more unused primers from the first amplification reaction; and a second amplification chamber for thermocycling to amplify one or more amplicons produced during the first amplification reaction by the addition of communal primers in a second amplification reaction, the amplicons of the first amplification reaction having at least one binding site for at least one communal primer.
• PCR chip comprising a first PCR chamber fluidly
• the second PCR chamber is fluidly connected to a hybridization and detection chamber, the hybridization and detection chamber comprising
• microspheres, or beads arranged so that the physical position of the beads is an indication of a specific target polynucleotide's presence in the sampled analyzed by means of the chip.
• the tem-PCR, and especially the arm-PCR, methods provide semiquantitative amplification of multiple polynucleotides in one reaction. Additionally, arm-PCR provides added sensitivity. Both provide the ability to amplify multiple polynucleotides in one reaction, which is beneficial in the present method because the repertoire of various T and B cells, for example, is so large.
• Clonal expansion due to recognition of antigen results in a larger population of cells which recognize that antigen, and evaluating cells by their relative numbers provides a method for determining whether an antigen exposure has influenced expansion of antibody-producing B cells or receptor-bearing T cells. This is helpful for evaluating whether there may be a particular population of cells that is prevalent in individuals who have been diagnosed with a particular disease, for example, and may be especially helpful in evaluating whether or not a vaccine has achieved the desired immune response in individuals to whom the vaccine has been given.
• 454A and 454B primers are linked onto PCR products either during PCR or ligated on after the PCR reaction.
• 454A and 454B primers may be used as communal primers in the amplification reactions.
• PCR products usually a mixture of different sequences, are diluted to about 200 copies per ⁇ .
• emulsion PCR (a semisolid gel like environment) the diluted PCR products are amplified by primers (454A or 454B) on the surface of the microbeads. Because the PCR templates are so dilute, usually only one bead is adjacent to one template, and confined in the semisolid
• a fiber-optic detector may be used to read the sequencing reaction from each well and the data is collected in parallel by a computer.
• One such high throughput reaction could generate up to 60 million reads (60 million beads) and each read can generate about 300bp sequences.
• One aspect of the invention involves the development of a database of
• epidemiological database that will produce valuable information, particularly in regard to the development of those diseases, such as cancer and heart disease, which are thought to often arise from exposure to viral or other infectious agents or transformed cells, many of which have as yet been unidentified.
• One particularly important use for the method of the invention involves the evaluation of children to determine whether infectious disease, environmental agents, or vaccines may be the cause of autism. For example, many have postulated that vaccine administration may trigger the development of autism.
• agents such as thimerosol in the vaccine, and studies have demonstrated that thimerosol does not appear to be a causative agent of the disease.
• cocktail vaccines has correlated with the rise in the number of cases of autism, however, gathering data to evaluate a potential causal connection for multiple antigens is extremely difficult.
• the method of the present invention simplifies that process and may provide key information for a better understanding of autism and other diseases in which the immune response of different individuals may provide an explanation for the differential development of disease in some individuals exposed to an agent or a group of agents, while others similarly exposed do not develop the disease.
• Imbalances of the immunoprofile may lead to many diseases, including cancers, leukemia, neuronal diseases (Alzheimer's, Multiple Sclerosis, Parkinson's, autism etc.), autoimmune diseases, and metabolic diseases. These diseases may be called immunoprofile diseases.
• immunoprofile diseases There may be two immunoprofile disease forms. (1 ) a “loss of function” form, and (2) a “gain of function” form.
• a “loss of function” form a person is susceptible to a disease because his/her restricted and/or limited immunoprofile lacks the cells that produce the most efficient and necessary IGs and TRs.
• gain of function a person is susceptible to a disease because his/her immunoprofile gained cells that produce IGs and TRs that normally should not be there.
• LEF loss of a function
• One aspect of the invention also provides a method comprising (a) amplifying and sequencing one or more RNAs from the T cells and/or B cells from one or more individuals, (b) inputting the sequences into a database to provide data which may be stored on a computer, server, or other electronic storage device, (c) inputting identifying information and characteristics for an individual corresponding to the sequences of the one or more RNAs as data which may also be stored on a computer, server, or other electronic storage device, and (d) evaluating the data of step (b) and step (e) for one or more individuals to determine whether a correlation exists between the one or more RNA sequences and one or more characteristics of the individual corresponding to the sequence(s).
• Identifying information may include, for example, a patient identification number, a code comprising the patient's HLA type, a disease code comprising one or more clinical diagnoses that may have been made, a "staging code" comprising the date of the sample, a cell type code comprising the type of cell subpopulation from which the RNA was amplified and sequenced, and one or more sequence codes comprising the sequences identified for the sample.
• the described method includes a novel primer design that not only allows amplification of the entire immunorepertoire, but also allows amplification in a highly multiplex fashion and semiquantitatively. Multiplex amplification requires that only a few PCR or RT-PCR reactions will be needed. For example, all IGs may be amplified in one reaction, or it could be divided into two or three reactions for IgH, IgL or IgK. Similarly, the T-cell receptors (TRs) may be amplified in just one reaction, or may be amplified in a few reactions including TRA, TRB, TRD, and TRG.
• TRs T-cell receptors
• Semi-quantitative amplification means that all the targets in the multiplex reaction will be amplified independently, so that the end point analysis of the amplified products will reflect the original internal ratio among the targets.
• the method can produce semi-quantitative amplification of polynucleotides comprising complementarity determining regions (CDRs), which result from genetic rearrangements within T or B cells and are responsible for the affinity and specificity of antibodies and/or T cell receptors for specific antigens.
• CDRs complementarity determining regions
• One aspect of the invention therefore relates to a method for analyzing semiquantitative sequence information to provide one or more immune status reports for a human or animal.
• the method for producing an immune status report comprising the steps of (a) identifying one or more distinct CDR3 sequences that are shared between a subject's immunoprofile and a disease library stored in a database, summing the total of those shared CDR3 sequences and computing the percentage of the total number of sequences in the subject's immunoprofile that are shared between the subject's immunoprofile and the disease library to create one or more original sharing indices; (b) randomly selecting sequences from a public library stored in a database to form a sub-library, the sub-library comprising a number of sequences that is approximately equal to the number of distinct sequences in the disease library, identifying one or more distinct CDR3 sequences that are shared between the subject's immunoprofile and the sub-library, summing the total of those shared CDR3 sequences and calculating the percentage of the total number of sequences in the subject's
• Figure 1 is a cartoon illustrating the difference that may be observed between, for example, the distinct type and number of T-cells present in a blood sample from a cancer patient (Fig. 1 a) and a healthy patient (Fig. 1 b), where each circle represents a distinct type of T-cell, as represented by an amplified and sequenced recombined cDNA of the
• Fig. 1 a indicates, there may be fewer distinct cells of different specificities, but larger numbers of cells of certain specificities, as represented by the CDR3 sequences.
• Fig. 1 b illustrates a normal profile of more different cells, but fewer numbers of each type of cell sharing the same CDR3 sequence.
• each distinct CDR3-expressing cell and the numbers of such cells represented within a blood or tissue sample from a human or animal, can constitute an immunoprofile for that human or animal.
• Compiling the immunoprofiles from a group of humans, for example, the group comprising both healthy individuals and individuals with various different diseases may provide a "public library" that is representative of the type of diversity found in a normal population (Fig. 2).
• compiling the immunoprofiles from a group of humans for example, the group comprising both healthy individuals and individuals with various different diseases may provide a "public library" that is representative of the type of diversity found in a normal population (Fig. 2).
• immunoprofiles of a group of individuals who have been clinically diagnosed with a particular disease may provide a "disease library" that is representative of the lack of diversity, the specific CDR3s of the expanded populations of cells, etc. (Fig. 3). These immunoprofiles may be stored in a database, accessible via computer access to the internet, for example, so that the information may be used in the method of the invention to analyze the immune status of a patient.
• An immunoprofile comprising a listing of distinct CDR3-expressing cells
• differentiate CDR3s those cells sharing a unique CDR3 sequence
• the numbers of each distinct CDR3 present in a blood or tissue sample from an individual may be produced for an individual patient.
• the patient's immunoprofile is compared to the combined immunoprofiles of a group of patients who have been diagnosed with a particular disease (a disease library, stored in a database). This can be done for a series of disease libraries, and shown in Fig. 4.
• immunoprofile as normal ("healthy") or characterized by the presence of a type and number of cells that have been associated with a particular disease.
• a patient supplies a clinical sample comprising, for example, blood or tissue, from which distinct CDR3s are semi-quantitatively amplified and sequenced. This provides the identity and the relative abundance of each CDR3 for all distinct CDR3s.
• This information may be entered into a program which accesses a database containing at least one public library and one or more disease libraries. Software used for data entry and/or analysis may be accessed via internet access to the database, or may be located on an individual personal computer, with internet access to the sequence information in the database. Comparisons are obtained between the individual immunoprofile and the various libraries and sub- libraries, and results are generated as generally illustrated in Fig. 4 and Fig.
• a P-value is calculated as the probability that a random percentage would be greater than or equal to the percentage noted for a particular disease library, and a significant result is noted when the fraction of times the sampling sharing indices exceeds the original sharing index for a particular library is less than 0.01 , for instance. If that sharing index represents the relationship between the individual's immunoprofile and a disease library, the individual may then be informed of the likelihood that the individual/patient has the disease represented by the specific disease library. If P- values computed against all disease libraries is greater than 0.01 , the individual's report may indicate that the immune profile looks normal and the disease state has not been detected.
• sequence data is compiled and stored in one or more databases for multiple populations of individuals, it may additionally be possible to associate certain sharing indexes with libraries representing populations with pre-conditions or predispositions to certain diseases.
• the immune system is both proactive and reactive, and changes in the immune system, reflected in the immunoprofile, may provide the first— and sometimes the only— signal that a predisposition, a precondition, or even an established disease is present.
• the inventors have utilized the method to demonstrate that certain types of cancers, inflammatory bowel disease, and certain viral infections may be detected by determining the sharing index between a patient and an established disease library, obtained by sequencing CDR3s using the ARM-PCR method to produce a subset of the immunorepertoire representing the CDR3s present.
• Blood samples may be taken from children prior to administration of any vaccines, those blood samples for each child establishing a "baseline" from which future samples may be evaluated.
• the future samples may be utilized to determine whether there has been an exposure to an agent which has expanded a population of cells known to be correlated with a disease, and this may serve as a "marker" for the risk of development of the disease in the future. Individuals so identified may then be more closely monitored so that early detection is possible, and any available treatment options may be provided at an earlier stage in the disease process.
• blood samples may be taken from children prior to administration of any vaccines, those blood samples from each child establishing a "baseline" from which future samples may be evaluated. For each child and for the entire population of children in the study, those baselines may be compared to the results of RNA sequencing of T and B cells using target-specific primers to amplify antibody and T-cell receptor, after vaccine administration. The comparison may further involve the evaluation of data regarding symptoms, diagnosed diseases, and other information associated for each individual with the corresponding antibody, and T- cell receptor sequences.
• the method of the invention may be especially useful for identifying commonalities between individuals with autoimmune diseases, for example, and may provide epidemiological data that will better describe the correlation between infectious and environmental factors and diseases such as heart disease, atherosclerosis, diabetes, and cancer— providing "biomarkers" that signal either the presence of a disease, or the tendency to develop disease.
• the method may also be useful for development passive immunity therapies.
• protective antibodies for example, and those antibodies may be utilized to provide passive immunity therapies in situations where such therapy is needed.
• the method of the invention may also provide the ability to accomplish targeted removal of cells with undesirable rearrangements, the method providing a means by which such cells rearrangements may be identified.
• T-cell-specific primers are shown in Table 1
• antibody- specific primers are shown in Table 2.
• An additional embodiment of the invention is a method of using any one or a combination of primers of Table 1 or Table 2, to amplify RNA from a blood sample, and more particularly to identify antibodies, T-cell receptors, and HLA molecules within a population of cells.
• Arm-PCR or tem-PCR may be used to amplify genes coding for the
• Primers are designated as F 0 (forward out), F, (forward in), R, (reverse in), R 0 (reverse out), FS (forward super primer) and RS, (reverse super primer), with super primers being common to a variety of the molecules due to the addition of a binding site for those primers at the end of a target-specific primer.
• the gene-specific primers (F 0 , F,, Ri, and R 0 ) are used at extremely low concentrations. Different primers are involved in the tem-PCR process at each of the three major stages. First, at the "enrichment" stage, low-concentration gene-specific primers are given enough time to find the templates.
• F 0 /R 0 For each intended target, depending on which primers are used, four possible products may be generated: F 0 /R 0 , F R 0 , F/R, and F 0 /R.
• the enrichment stage is typically carried out for 10 cycles.
• the annealing temperature is raised to 72°C, and only the long 40-nucleotide inside primers (F, and R) will work.
• F, and R the PCR products are "tagged” with the universal super primer sequences.
• high-concentration super primers work efficiently to amplify all targets and label the PCR products with biotin during the process.
• Specific probes may be covalently linked with Luminex color-coated beads.
• the inventor designed nested primers based on sequence information in the public domain. For studying B and T cell VDJ rearrangement, the inventor designed primers to amplify rearranged and expressed RNAs. Generally, a pair of nested forward primers is designed from the V genes and a set of reverse nested primers are designed from the J or C genes. The average amplicon size is 250-350bp. For the IgHV genes, for example, there are 123 genes that can be classified into 7 different families, and the present primers are designed to be family specific. However, if sequencing the amplified cDNA sequences, there are enough sequence diversities to allow further differentiation among the gene within the same family. For the MHC gene locus, the intent is to amplify genomic DNA. EXAMPLES
• each CDR3 has its own frequency Si , s 2 , ... s n .
• D is a disease library, which is the sum of a certain number of patients'
• P is a public library, which is the sum of a large number of control's
• the Sharingjndex is defined as the sum of s x , s y , ... s z , where CDR3 X ,
• CDR3 y , ... CDR3 Z are shared in the subject's immunoprofile and a library. Note that s x , s y , ... s z is the frequency of CDR3s in the subject's immunoprofile, not in the library.
• M unique CDR3s in the public library are randomly selected and used to create a sub-library P1 and the sharing index (Sl p ) between the subject and the sub-library computed according to above formula.
• the sampling procedure is repeated 1000 or more times and 1000 or more Sl px are computed .
• the P-value is defined as the fraction of all Sis (Sl p i , SI P 2, ... Sl pX, Sl d, (Note that Sl d is included), which is equal to or greater than Sl d . Note that, when sampling CDR3s in the public library, CDR3s found in x control's immunoprofiles are given x times of chances to be sampled. Amplification of T or B Cell Rearrangement Sites
• All oligos were resuspended using 1x TE. All oligos except 454A and 454B were resuspended to a concentration of I OOpmol/ ⁇ -. 454A and 454B were resuspended to a concentration of I OOOpmol/ ⁇ - 454A and 454B are functionally the same as the communal primers described previously, the different sequences were used for follow up high throughput sequencing procedures.
• An Alpha Delta primer mix included
• primers (all of TRAV-C + TRDV-C), a Beta Gamma primer mix included 79 primers (all of TRBVC and TRGV-C) and a B cell primer mix that included a total of 70 primers.
• F 0 , F,, and R, primers were at a concentration of 1 ⁇ / ⁇ _.
• R 0 primers were at a concentration of 5 pmol/ ⁇ -.
• 454A and 454B were at a concentration of 30 pmol/ ⁇ -.
• RT-PCR was performed using a Qiagen One-Step RT-PCR kit. Each sampl contained the following:
• the initial experiment showed that a smear is generated from PCR reactions where templates were included.
• the smears indicate different sizes of PCR products were generated that represented a mixture of different VDJ rearrangements. There is some background amplification from the B cell reaction. Further improvement on that primer mix was required to clean up the reaction.
• PCR products generated from the Alpha Delta mix and the Beta Gamma mix (lanes 2 and 3 in Fig. 1 a) were diluted 1 :1000 and a 2 ⁇ aliquot used as PCR template in the following reaction. Then, instead of using a mixture of primers that targeting the entire repertoire, one pair of specific Fi and Ri primers were used (5 pmol each) to amplify only one specific PCR product. The following cycling conditions were used to amplify the samples:
• a Qiagen PCR kit was used to amplify the products.
• the Master Mix used for the PCR contained the following:
• the photograph of the gel in Fig. 1 b shows the PCR products of the following reactions: (1 ) Ladder; (2) TRAV1 Fi+TRACRi with alpha delta Pan T PCR product; (3) TRAV2Fi+TRACRi with alpha delta Pan T PCR product; (4) TRAV3F
• TRAV1 Fi+TRACRi with alpha delta Pan T PCR product (8) TRAV2Fi+TRACRi with alpha delta Pan T PCR product; (9) TRAV3Fi+TRAC with alpha delta Pan T PCR product; (10) TRAV4Fi+TRACRi with alpha delta Pan T PCR product; (1 1 )
• TRAV5Fi+TRACRi with alpha delta Pan T PCR product; (12) PCR Blank.
• Primers listed as F-i are "forward inner” primers and primers listed as F 0 are “forward outer” primers, with Ri and R 0 indicating “reverse inner” and “reverse outer” primers, respectively.
• Fig. 1 b a single PCR product was generated from each reaction. Different size bands were generated from different reactions.
• PCR templates used in this reaction were diluted PCR products (1 :1000) of previous reactions that used primer mixes to amplify all possible VDJ rearrangements (for example, a primer mix was used that included total of 82 primers to amplify T cell receptor Alpha and Delta genes) and (2) Only one pair of PCR primers, targeting a specific V gene, are used in each reaction during this "cloning" experiment. Some of these products were gel purified and sequenced. The following are example sequences obtained from the protocol described above. In every case, a single clone was obtained, and a specific T cell receptor V gene that matched the Fi primer was identified.
• TCR3 transcriptome the inventors conducted control experiments using chemically synthesized TCR3 CDR3 templates. For this, the inventors chemically synthesized four distinct clones, clonally purified each clone, and prepared different mixes of the four constructs as templates for amplicon rescue multiplex (ARM)-PCR. Two different reaction mixtures were subjected to two independent ARM-PCR reactions, and the pooled PCR products were sequenced at a length of 100bp from both ends using the lllimuna HiSeq2000®. The inventors first joined together paired-end reads through overlapping alignment with a modified Needleman-Wunsch algorithm, and then mapped the merged sequences to germline V, D and J reference sequences.
• ARM amplicon rescue multiplex
• template mix I a total of 29,804 sequences, which corresponded to 609 unique CDR3 variants, were removed.
• template mix II 54,516 artifactual sequences (831 unique CDR3 variants) were identified.
• the reference filter is ineffective at the V-J and D-J junctions because the randomly added nucleotides in these regions during somatic recombination cannot be mapped. Therefore, the inventors implemented a PCR filter after computational simulation experiments to better understand four variables: the impact of the initial template number, the replication efficiency of each cycle, the cycle number (n), and the DNA polymerase error rate ( ⁇ ) on the total end-point error rate. In contrast, the inventors noted that the PCR polymerase error rate has a pronounced effect on the number of accumulated errors
• the PCR efficiency was set to decreased 5% per cycle for the first 25 cycles and 10% per cycle for the remaining cycles. The PCR efficiency was reset to 1.0 for each fresh PCR reaction. Furthermore, the inventors allowed mutation at the second position. Published substitution error rates for Taq enzyme, expressed as errors per bp per cycle, range from 0.023 xlO "4 to 2.1 xl O "4 . In the simulation experiments, the substitution error rate was set at 2.7 x10 "5 , and the insertion-deletion (indel) error rate was set as 1.0 x10 "6 . Taq polymerase is known to have a much higher insertion-and- deletion (indel) mutation rate in homopolymeric region of templates. For a
• the indel error rate in a homopolymeric region was set as n x ⁇ , where n is the length of the homopolymeric region and ⁇ is 1.0 x 10 "6 .
• the inventors developed a computational "mosaic filter.” Using this filtering algorithm, the inventors identified a total of 17 and 15 chimeric sequences in template mixtures I and II, respectively. Of note, some of these CDR3 chimeras displayed sequence copy numbers >1000, indicating that the inventors' algorithm for the filter is capable of identifying high- abundance chimeric CDR3 sequences.
• TRAVI 6F1 TCTAGAGAGA 55 GCCTCCCTCGC 56
• TRAVI 8F1 CAGGAGACG 61 GCCTCCCTCGC 62
• TRBV-C TRBV1 Fo AATGAAACGT 120 AATGAAACGTG 120
• TRBV1 1 FO CCTAAGGATC 158 CCTAAGGATCG 158 Locus Primer Sequence SEQ ID Sequence SEQ ID Name NO. NO.
• TRBV1 1 Fi ACTCTCAAGA 159 GCCTCCCTCGC 160
• TRBV13Fo CTATCCTATC 166 CTATCCTATCC 166
• TRBVI 6F1 AATGTCTTTG 176 GCCTCCCTCGC 177
• TRBVI 8F1 AATATCATAG 182 GCCTCCCTCGC 183
• TRBV27Fo TTGTTCTCAG 205 TTGTTCTCAGA 205
• TRBV28Fo ATGTGTCCAG 208 ATGTGTCCAGG 208
• TRBCRi TCTGATGGCT 218 GCCTTGCCAGC 219
• TRGV-C TRGV1-5Fo GGGTCATCTG 232 GGGTCATCTGC 232
• TRGV1 1 FO AGAGTGCCCA 244 AGAGTGCCCAC 244
• TRGV1 1 Fi GCTCAAGATT 245 GCCTCCCTCGC 246
• GAGTCAC CCATCAGAGTTC CAGGGCAGAGTC AC
• GAGTCAC CCATCAGAGTTC CAGGAAAGAGTC AC
• GAGTCAC CCATCAGAATTC CAGGACAGAGTC AC
• IgKV-C IgKVI Fo TAGGAGACAGAG 302 TAGGAGACAGAG 302
• CAGAAACC CAGAAACC

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• 2014
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