BACKGROUND
activation of the immune system, together with genomic alternation, including somatic hypermutation and recombination, establishes innate and adaptive immunity in the immune system  <cit> .
both the immunoglobulin  and the t cell receptor  loci contain many different v, d and j segments, which are subject to a tightly regulated genomic rearrangement process – vj recombination – during early lymphoid differentiation  <cit> .
for a given tcr subtype, complementarity determining region  <dig>  is generated by the vj combination at this subtype locus, with the translated protein sequence forming the center of the antigen binding site  <cit> , defining the affinity and specificity of the receptor for individual peptide-mhc complexes.
the cdr <dig> sequence of dominant clones in cancer patient may serve as a signature to diagnose cancer or to classify tumor into subtypes.
in addition, signatures could be obtained at the time of disease diagnosis and then monitored on an ongoing basis, to assess the effects of anticancer therapies or for early detection of recurrence  <cit> .
the importance of monitoring tcr in human health and disease has been increasingly recognized.
recent studies showed that tcr repertoire has been found to affect a wide range of diseases, including malignancy, autoimmune disorders and infectious diseases, and, given the broad involvement of the immune system in almost all of human health and disease, this reach should be expected to expand greatly  <cit> .
however, conventional methods to measure vj recombination have several limitations to prevent detailed characterization of immune repertoire.
more recent approaches, such as multiparameter flow cytometry, spectrotyping, or custom-designed real-time pcr assays, are slightly more quantitative and offer higher resolution, but these methods are labor intensive and are unable to offer sequence-level insights as to the exact vj recombination patterns in patients.
solving this problem will enable the wider application of monitoring immune repertoire in clinical settings.
with the development of massively parallel, single-molecule sequencing techniques, it has now become feasible to assay vj recombination by next-generation sequencing, as a means to exhaustively profile the immune repertoire within human subjects.
for example, the roche  <dig> sequencers has been used to measure and clinically monitor human lymphocyte clonality  <cit> , which takes advantage of 454’s ability to generate longer sequencing reads that potentially covers vj recombination junction points.
another similar study also used roche  <dig> to study human t cell subsets  <cit> .
note that they separated t cell subpopulations and focused on tcr loci only.
however, other investigators have focused on illumina genome analyzer or hiseq that generates only ~ <dig> bp to ~ <dig> bp reads.
for example, a group has developed a short reads assembly strategy to first assemble  <dig> bp sequences and then sample the cdr <dig> diversity in human t lymphocytes from peripheral blood  <cit> .
the data analysis involved in such strategy is much less straightforward, but the technology is more accessible and cost-effective in large scale studies.
several additional tcr-seq studies  <cit>  have been extensively reviewed recently.
some of the commonalities include:  they almost always used t-cell dna or rna as the first starting material;  most of the studies use multiplex pcr reactions to enrich the vj recombinations for next-generation sequencing;  large-scale t-cell repertoire analysis has been limited to interrogation of a single tcr subunit per sequencing run, although functional antigen-engaging tcrs are heterodimeric proteins comprising both an α and a ß chain.
in the current study, we explored the feasibility of using ligation-anchored pcr reaction, rather than multiplex pcr reactions, for unbiased amplification of tcr-beta  repertoire.
the key aspect of our approach is ligating a synthesized linker oligo sequence  to the 3’end of single-stranded cdna, thus it is applicable to extracted rna samples.
additionally, similar to our previous study examining immunoglobulin repertoire  <cit> , instead of relying on flow cytometry or magnetic beads to isolate t cells or b cell populations from peripheral blood, we attempted to assay rnas extracted from whole blood directly.
finally, we evaluated sequencing data from the latest illumina hi-seq <dig> sequencer, which can generate  <dig> bp single-end reads , with a significantly shortened turnaround time  compared to illumina hi-seq <dig> or roche  <dig> sequencer.
despite the shorter reads than  <dig> sequencer or illumina miseq sequencer,  <dig> bp read length is still long enough to map vj recombination sites, given appropriate primer design for pcr amplification.
to evaluate this approach, we did a pilot study on patients suffered from meningiomas, the most common primary brain tumors in the united states  <cit> , and compared differences of the detected trb repertoire in patients with benign  and malignant  tumors to healthy individuals.
this tumor arises from the membranous layers surrounding the central nervous system , and is not subject to blood-brain barrier.
several previous studies have reported the presence of both humoral  <cit>  and cellular  <cit>  immune responses in patients with meningiomas.
indeed, it has been proposed that frequent antibody response against specific antigens in even benign meningioma can serve as diagnostic targets  <cit> .
the immune response to meningiomas could be similar to the reported expansion of tumor infiltrating lymphocytes in response to cancerous cells  <cit> .
therefore, in addition to testing the technical feasibility, our pilot study has the added value of investigating whether there is increased clonality with increased severity of cancer, and whether different patients have similar or distinct cdr <dig> sequences in clonal t cells.
RESULTS
total rna extraction and pcr amplification of trb variable region
to explore all the actively transcribed immune receptor variable regions within whole blood in an unbiased manner, we developed an integrative approach to extract total rna from whole blood, followed by a pcr protocol to capture the variable region  of specific immune receptor .
in this scheme, after total rna extraction from whole blood, we generate single-stranded cdna from reverse transcription of mrna.
after ligation of a synthesized linker oligo sequence to the 3’end of single-stranded cdna, pcr reaction was carried out using a primer pair  to target the linker sequence and the consensus starting sequence of immune receptor constant region , respectively.
the p-vdj primer was specifically designed such that it did not have high sequence homology to any other region other than the tcr locus, and that it avoids any known snps.
in our proof-of-concept study, total rna extracted from whole blood of a healthy donor was used to test our methodology on capturing trb variable region .
for samples that were subject to both reverse transcription  and linker ligation, pcr reaction resulted in a predominant band with fragment size around  <dig> bp, roughly the size expected for trb variable region.
for control samples without reverse transcription, this band was not visible.
additionally, controls without linker ligation did not produce this specific band in dna gel.
thus, the protocol developed in our study should be able to capture transcribed trb variants from whole blood directly, without the need to separate t cells first.figure  <dig> pcr method utilizing single target-specific primer to explore actively transcribed immune genes.
a), the scheme of an integrative approach to explore immune repertoire from whole blood ; b), dna gel image of pcr amplified variable region  of trb gene from a healthy donor under different conditions; c), dna gel image of amplicons from malignant and benign patients under linker ligation or control condition.
trb profiling using illumina high-throughput sequencing
to further evaluate whether the developed protocol is applicable to profile trb repertoire of clinical samples, we tested the protocol on several blood samples from patients with malignant and benign meningiomas .
the fact that immune responses are present in patients with meningiomas  <cit>  suggests that immune receptors such as tcr may respond to this tumor type and serve as a biomarker for diagnosis.
we followed the same protocol to profile trb v-region variants using previously collected whole blood samples  from two malignant patients  and two benign patients , respectively.
compared to controls without linker ligation, all four reverse transcribed samples with ligation showed the expected predominant band , suggesting the robustness of this approach.
the amplified fragments were further used for next-generation sequencing library preparation.
as a control, biological duplicate libraries from two healthy donors  were also constructed for illumina sequencing.
purified pcr amplicons were barcoded with illumina truseq kit for high-throughput sequencing.
the latest illumina hiseq- <dig> sequencer was used for sequencing, with load of ~30% phix for proper matrixing and phasing into the lane.
collected sequencing data was filtered, and the reads with either p-u or p-vdj end tag at the 5’end were selected and mapped using igblast software .
reproducibility of the method was measured with pearson’s correlation for the mapped reads from biological replicate data, and plotted using density scatterplots .
in general, the correlation coefficients were between  <dig>  and  <dig>  for the biological replicates, suggesting a fair consistency of the data.
enrichment of trb fragments and usage of trb v-segments
after mapping to germline tcr v gene database using igblast, a significant portion of the reads with either p-u or p-vdj end tag contained trb fragments .
however, we observed a general higher mapping rate for every p-vdj tagged pool compared to p-u pool from the same dataset.
it is possible that higher noise signals from p-u tagged reads were from p-u linker ligation step that the linker sequence can be attached to the 3’end of any template single stranded cdna molecule.
as note, for either p-u or p-vdj tagged reads, 64% to 96% of the mapped tcrv-containing reads  belong to trb subtype.
therefore, ligation-anchored pcr approach is specific to profile trb repertoire from blood samples.figure  <dig> specificity of the integrative approach on profiling v-region of trb and v-segment usage.
a &b), subtype population of sequenced reads that are mappable to v-region of tcr ; c) v-segment usage from vj-containing reads that are translationally productive .
we next compared the usage of different v-segments from translationally productive vj reads within p-vdj tagged read pools  from patients with meningiomas and from healthy donors .
we noticed a subset of trb v-segments with increased usage in meningiomas.
v-segments trbv <dig>  trbv6- <dig> and trbv7- <dig> showed highest usage in both malignant 010_m and 221_m samples.
specific to individual malignant patient, 010_m showed drastic increase of trbv10- <dig>  trbv <dig>  trbv24- <dig> and trbv <dig>  while 221_m had elevated levels of trbv <dig> and trbv4- <dig> levels.
also noted, trbv7- <dig> showed increase of usage in both benign samples, while v12- <dig>  v5- <dig> and v7- <dig> levels were greatly increased in either benign individual.
therefore, the statistics of v-segment usage revealed the potential expansion of immune cells with particular trbv subtypes, possibly reflecting human immune response to meningiomas.
exploration of v-j segment pairing in vj recombination
our method utilized a p-vdj primer to read through vj junction sequences from the reverse complement strand of the 5’-end common sequence of trbc <dig> and trbc <dig>  with  <dig> bp read length, the reads can extend to fr <dig> region of v-segment for most of the vj recombination events.
to explore v-j pairing in vj recombination, we counted and normalized pairing frequencies between different v- and j-segments, and presented the results in circos plots  <cit>  .
interesting to note, pairing of trbv7- <dig> with trbj <dig>  occurred more frequently in both malignant meningiomas samples  compared to benign ones , and almost abolished in normal controls .
in contrast, pairing of trbv5- <dig> or trbv7- <dig> with trbj <dig>  was enriched in normal controls but not in patient samples.
however, in general, the v-j pairing patterns shown with circos plots appear to be distinct for each patient.
the 5-way overlap of v-j pairing events of individual patient with normal control pool was presented in chow-ruskey diagram .
as shown in figure 3b, the events detected in normal pool is largely different from those in the patients, with less than half of the v-j pairing overlapped with any patient sample.
in addition, a number of events detected in individual patient sample are unique v-j pairing.
also noted, v-j pairing detected in individual malignant patient was less than the pairing in benign or normal samples.
in summary, from v-j pairing analysis, it is reasonable to postulate that particular subtype of vj recombinant t cells expand dramatically in patient blood, thus relative weight of other subtype t-cells in the total population decreases accordingly.figure  <dig> v-j pairing events identified from p-vdj tagged reads.
a), circos maps illustrating pairing frequencies of v-segments and j-segments from vj-containing reads ; b), chow-ruskey diagram showing the five-way overlap of v-j pairing events in 010_m, 221_m, 347_b, 392_b with normal pool.
characterization of “immune signature” – cdr3
in immune system, specific t cell clone can expand dramatically in response to tumor antigens.
counting analysis of v-j pairing  showed less number of pairing in malignant meningiomas  compared to benign meningiomas  or normal controls .
but when we counted v-j pairing at a cutoff of 5% of total population, we found more pairing events in malignant  than benign  or normal .we then asked whether significantly expanded v-j pairing events in malignant patients could result from clonal expansion of particular t cells in which the sequence of cdr <dig> region can serve as a signature.
we chose the v-j pairing events that are greatly expanded in either malignant individual  compared to normal controls  for cdr <dig> sequence analysis.
in total, eight pairing events trbv6- <dig> with trbj1- <dig>  trbv <dig> with trbj1- <dig>  trbv3- <dig> with trbj2- <dig>  trbv24- <dig> with trbj1- <dig>  trbv <dig> with trbj2- <dig>  trbv29- <dig> with trbj1- <dig>  trbv7- <dig> with trbj1- <dig>  and trbv4- <dig> with trbj2- <dig> were identified as dramatically increased in either one of the malignant samples.
pairing of trbv7- <dig> and trbj <dig>  that showed moderate increase in both malignant samples  was also included for cdr <dig> analysis.
except for pairing of trbv3- <dig> with trbj2- <dig> which has unusual cdr <dig> skipping, sequencing reads for every other combination showed one dominant fr3-cdr <dig> junction dna sequence , which accounts for 87% to 95% of the corresponding pairing event, suggesting clonal expansion.
we also generated protein sequence logos for further comparison.
in protein sequence logo plots, the amino acid sequences between the conserved cys  at the 3’end of fr <dig> region  and the common vfppey motif at the 5’end of trb constant region varied a lot for different v-j pairing events.
in malignant meningiomas, both trbc <dig> and trbc <dig> subtypes were used in those expanded pairing events.
as expected, most of the j subtypes  fused to the closet known trbc subtypes .
however, the dominant clone for trbv24- <dig> with trbj1- <dig> produced an unusual recombination/mutation at the beginning part of trb constant region that is not mappable to either trbc <dig> or trbc <dig>  for pairing of trbv7- <dig> with trbj2- <dig> that showed expansion in both malignant samples, we observed that 88% to 90% of this population was from single dominant cdr <dig> signature.
however, their translated cdr <dig> sequences were not identical , suggesting occurrence of other genomic editing events, such as hypermutation.
in summary, cdr <dig> sequence logo analysis identified cdr <dig> signature sequences associated with individual malignant patient, which may reflect expansion of several specific v-j pairing clones in patient blood.figure  <dig> sequence logos for detected fr3- trbc portions of malignant meningiomas.
visualized in the dna sequence logos are the dominant clonal cdr <dig> sequences of selected v-j pairings ; the translated protein sequence logos illustrate antigen recognition regions from the end of fr <dig> and the beginning of trbc.
trbc <dig> and trbc <dig> sequences are underlined in purple and cyan colors, respectively.
discussion and
CONCLUSIONS
in the current study, we presented an integrated approach by using single primer pcr together with next-generation sequencing to interrogate immune repertoire of tcr-beta.
we have demonstrated the technical feasibility to use this system to infer immune repertoire, using whole blood from four meningiomas patients and two healthy donors.
by aligning reads to a sequence database of germline v-genes, d-genes and j-genes, the usage of different v-gene segments was quantified.
interestingly, comparison between malignant, benign and normal groups identified an increased usage of trbv <dig>  trbv6- <dig> and trbv7- <dig> in malignant meningiomas.
however, the pairing of v-j subtypes for recombination revealed a generally diversified immune repertoire for individual patient, although trbv7- <dig> with trbj <dig>  appears to be associated with malignant transformation.
further analysis of cdr <dig> region sequence logos of the top expanded v-j pairing in malignant meningiomas indicated distinct cdr <dig> signatures for the two malignant patients.
however, we caution that these observations were made on a small number of samples, and they may not have any biological significance.
our purpose is to use these data to demonstrate the technical feasibility of “single-primer” interrogation of immune repertoire, rather than determining what differs between malignant and benign tumors.
there are several unique aspects of our protocol, compared to previous studies.
first of all, total rna is extracted directly from frozen blood samples for profiling, thus the procedure can be easily adapted for clinical application.
second, by using ligation-anchored pcr for amplification, all the recombination events at a particular immune gene locus is likely to be amplified in an unbiased manner.
furthermore, sequencing of barcoded libraries through illumina hi-seq  <dig> ensures fast turn-around time  and good sequencing depth  at a relatively low cost.
finally, we recognize that more recent generations of illumina sequencers can now sequence  <dig> bp or even continuous  <dig> bp reads, potentially further reduce the computational complexity and increase the rate of recovering full length vj recombination for our approach.
there are several major limitations of our protocol as well.
first, due to the need to add in 3’-adaptors to the cdna terminus for ligation-anchored pcr, our method relies on rna samples, which are less readily available and more vulnerable to degradation, compared to genomic dna samples.
however, in the current study, we used frozen whole blood samples and still obtained satisfactory results, suggesting that it is practically feasible to use this method in real-world clinical settings.
second, although illumina hi-seq  <dig> provided longer read length  than illumina hi-seq  <dig>  to cover immune signature region cdr <dig>  longer read length is still needed to cover the entire variable region of immune genes.
thus the latest illumina miseq with  <dig> bp or 2x <dig> bp chemistry should be more suitable for profiling immune repertoire.
third, our sequencing data showed higher sequencing depth of malignant sample libraries  compared to benign ones .
we acknowledge that uneven mixing of the constructed libraries for sequencing could be the reason.
to address the issue of sequencing depth, we performed additional data analysis using the first  <dig> million sequencing reads of 010_m and 221_m data .
of note, the results from the 7-million-read data were consistent with the conclusions from the whole sequencing data, indicating that the sequencing depth in our study is deep enough.
fourth, we specifically designed primers to target tcr-beta isoforms.
our sequencing data contained some other tcr isoforms as well, suggesting the presence of some minor cross reactions.
our future studies may design specific primers for other types of isoforms as well, to investigate whether it is feasible to interrogate all major tcr isoforms in the same sequencing run.
finally, the sample size in our study is small, which prevents us to draw any statistically significant conclusions on clonal expansion of trb subtypes associated with malignant transformation of meningiomas; however, the results from high-throughput sequencing data analysis do support validity of the developed integrated approach.
in conclusion, we have developed a ligation-anchored pcr approach to interrogate immune repertoire, and demonstrated its technical feasibility and effectiveness in capturing the tcr-beta landscapes.
recently, human antibody repertoire has been reported to change in response to influenza vaccination  <cit> .
we expect further development of our technology may enable a comprehensive delineation of immune repertoire, including other forms of tcrs as well as immunoglobulins.

 activation of the immune system, together with genomic alternation, including somatic hypermutation and recombination, establishes innate and adaptive immunity in the immune system. both the immunoglobulin and the t cell receptor loci contain many different v, d and j segments, which are subject to a tightly regulated genomic rearrangement process – vj recombination – during early lymphoid differentiation. for a given tcr subtype, complementarity determining region is generated by the vj combination at this subtype locus, with the translated protein sequence forming the center of the antigen binding site, defining the affinity and specificity of the receptor for individual peptide-mhc complexes. the cdr sequence of dominant clones in cancer patient may serve as a signature to diagnose cancer or to classify tumor into subtypes. in addition, signatures could be obtained at the time of disease diagnosis and then monitored on an ongoing basis, to assess the effects of anticancer therapies or for early detection of recurrence. the importance of monitoring tcr in human health and disease has been increasingly recognized. recent studies showed that tcr repertoire has been found to affect a wide range of diseases, including malignancy, autoimmune disorders and infectious diseases, and, given the broad involvement of the immune system in almost all of human health and disease, this reach should be expected to expand greatly. however, conventional methods to measure vj recombination have several limitations to prevent detailed characterization of immune repertoire. more recent approaches, such as multiparameter flow cytometry, spectrotyping, or custom-designed real-time pcr assays, are slightly more quantitative and offer higher resolution, but these methods are labor intensive and are unable to offer sequence-level insights as to the exact vj recombination patterns in patients. solving this problem will enable the wider application of monitoring immune repertoire in clinical settings. with the development of massively parallel, single-molecule sequencing techniques, it has now become feasible to assay vj recombination by next-generation sequencing, as a means to exhaustively profile the immune repertoire within human subjects. for example, the roche sequencers has been used to measure and clinically monitor human lymphocyte clonality, which takes advantage of 454’s ability to generate longer sequencing reads that potentially covers vj recombination junction points. another similar study also used roche to study human t cell subsets. note that they separated t cell subpopulations and focused on tcr loci only. however, other investigators have focused on illumina genome analyzer or hiseq that generates only ~ bp to ~ bp reads. for example, a group has developed a short reads assembly strategy to first assemble bp sequences and then sample the cdr diversity in human t lymphocytes from peripheral blood. the data analysis involved in such strategy is much less straightforward, but the technology is more accessible and cost-effective in large scale studies. several additional tcr-seq studies have been extensively reviewed recently. some of the commonalities include: they almost always used t-cell dna or rna as the first starting material; most of the studies use multiplex pcr reactions to enrich the vj recombinations for next-generation sequencing; large-scale t-cell repertoire analysis has been limited to interrogation of a single tcr subunit per sequencing run, although functional antigen-engaging tcrs are heterodimeric proteins comprising both an α and a ß chain. in the current study, we explored the feasibility of using ligation-anchored pcr reaction, rather than multiplex pcr reactions, for unbiased amplification of tcr-beta repertoire. the key aspect of our approach is ligating a synthesized linker oligo sequence to the 3’end of single-stranded cdna, thus it is applicable to extracted rna samples. additionally, similar to our previous study examining immunoglobulin repertoire, instead of relying on flow cytometry or magnetic beads to isolate t cells or b cell populations from peripheral blood, we attempted to assay rnas extracted from whole blood directly. finally, we evaluated sequencing data from the latest illumina hi-seq sequencer, which can generate bp single-end reads, with a significantly shortened turnaround time compared to illumina hi-seq or roche sequencer. despite the shorter reads than sequencer or illumina miseq sequencer, bp read length is still long enough to map vj recombination sites, given appropriate primer design for pcr amplification. to evaluate this approach, we did a pilot study on patients suffered from meningiomas, the most common primary brain tumors in the united states, and compared differences of the detected trb repertoire in patients with benign and malignant tumors to healthy individuals. this tumor arises from the membranous layers surrounding the central nervous system, and is not subject to blood-brain barrier. several previous studies have reported the presence of both humoral and cellular immune responses in patients with meningiomas. indeed, it has been proposed that frequent antibody response against specific antigens in even benign meningioma can serve as diagnostic targets. the immune response to meningiomas could be similar to the reported expansion of tumor infiltrating lymphocytes in response to cancerous cells. therefore, in addition to testing the technical feasibility, our pilot study has the added value of investigating whether there is increased clonality with increased severity of cancer, and whether different patients have similar or distinct cdr sequences in clonal t cells.  total rna extraction and pcr amplification of trb variable region to explore all the actively transcribed immune receptor variable regions within whole blood in an unbiased manner, we developed an integrative approach to extract total rna from whole blood, followed by a pcr protocol to capture the variable region of specific immune receptor. in this scheme, after total rna extraction from whole blood, we generate single-stranded cdna from reverse transcription of mrna. after ligation of a synthesized linker oligo sequence to the 3’end of single-stranded cdna, pcr reaction was carried out using a primer pair to target the linker sequence and the consensus starting sequence of immune receptor constant region, respectively. the p-vdj primer was specifically designed such that it did not have high sequence homology to any other region other than the tcr locus, and that it avoids any known snps. in our proof-of-concept study, total rna extracted from whole blood of a healthy donor was used to test our methodology on capturing trb variable region. for samples that were subject to both reverse transcription and linker ligation, pcr reaction resulted in a predominant band with fragment size around bp, roughly the size expected for trb variable region. for control samples without reverse transcription, this band was not visible. additionally, controls without linker ligation did not produce this specific band in dna gel. thus, the protocol developed in our study should be able to capture transcribed trb variants from whole blood directly, without the need to separate t cells first.figure pcr method utilizing single target-specific primer to explore actively transcribed immune genes. a), the scheme of an integrative approach to explore immune repertoire from whole blood; b), dna gel image of pcr amplified variable region of trb gene from a healthy donor under different conditions; c), dna gel image of amplicons from malignant and benign patients under linker ligation or control condition. trb profiling using illumina high-throughput sequencing to further evaluate whether the developed protocol is applicable to profile trb repertoire of clinical samples, we tested the protocol on several blood samples from patients with malignant and benign meningiomas. the fact that immune responses are present in patients with meningiomas suggests that immune receptors such as tcr may respond to this tumor type and serve as a biomarker for diagnosis. we followed the same protocol to profile trb v-region variants using previously collected whole blood samples from two malignant patients and two benign patients, respectively. compared to controls without linker ligation, all four reverse transcribed samples with ligation showed the expected predominant band, suggesting the robustness of this approach. the amplified fragments were further used for next-generation sequencing library preparation. as a control, biological duplicate libraries from two healthy donors were also constructed for illumina sequencing. purified pcr amplicons were barcoded with illumina truseq kit for high-throughput sequencing. the latest illumina hiseq- sequencer was used for sequencing, with load of ~30% phix for proper matrixing and phasing into the lane. collected sequencing data was filtered, and the reads with either p-u or p-vdj end tag at the 5’end were selected and mapped using igblast software. reproducibility of the method was measured with pearson’s correlation for the mapped reads from biological replicate data, and plotted using density scatterplots. in general, the correlation coefficients were between and for the biological replicates, suggesting a fair consistency of the data. enrichment of trb fragments and usage of trb v-segments after mapping to germline tcr v gene database using igblast, a significant portion of the reads with either p-u or p-vdj end tag contained trb fragments. however, we observed a general higher mapping rate for every p-vdj tagged pool compared to p-u pool from the same dataset. it is possible that higher noise signals from p-u tagged reads were from p-u linker ligation step that the linker sequence can be attached to the 3’end of any template single stranded cdna molecule. as note, for either p-u or p-vdj tagged reads, 64% to 96% of the mapped tcrv-containing reads belong to trb subtype. therefore, ligation-anchored pcr approach is specific to profile trb repertoire from blood samples.figure specificity of the integrative approach on profiling v-region of trb and v-segment usage. a &b), subtype population of sequenced reads that are mappable to v-region of tcr; c) v-segment usage from vj-containing reads that are translationally productive. we next compared the usage of different v-segments from translationally productive vj reads within p-vdj tagged read pools from patients with meningiomas and from healthy donors. we noticed a subset of trb v-segments with increased usage in meningiomas. v-segments trbv trbv6- and trbv7- showed highest usage in both malignant 010_m and 221_m samples. specific to individual malignant patient, 010_m showed drastic increase of trbv10- trbv trbv24- and trbv while 221_m had elevated levels of trbv and trbv4- levels. also noted, trbv7- showed increase of usage in both benign samples, while v12- v5- and v7- levels were greatly increased in either benign individual. therefore, the statistics of v-segment usage revealed the potential expansion of immune cells with particular trbv subtypes, possibly reflecting human immune response to meningiomas. exploration of v-j segment pairing in vj recombination our method utilized a p-vdj primer to read through vj junction sequences from the reverse complement strand of the 5’-end common sequence of trbc and trbc with bp read length, the reads can extend to fr region of v-segment for most of the vj recombination events. to explore v-j pairing in vj recombination, we counted and normalized pairing frequencies between different v- and j-segments, and presented the results in circos plots. interesting to note, pairing of trbv7- with trbj occurred more frequently in both malignant meningiomas samples compared to benign ones, and almost abolished in normal controls. in contrast, pairing of trbv5- or trbv7- with trbj was enriched in normal controls but not in patient samples. however, in general, the v-j pairing patterns shown with circos plots appear to be distinct for each patient. the 5-way overlap of v-j pairing events of individual patient with normal control pool was presented in chow-ruskey diagram. as shown in figure 3b, the events detected in normal pool is largely different from those in the patients, with less than half of the v-j pairing overlapped with any patient sample. in addition, a number of events detected in individual patient sample are unique v-j pairing. also noted, v-j pairing detected in individual malignant patient was less than the pairing in benign or normal samples. in summary, from v-j pairing analysis, it is reasonable to postulate that particular subtype of vj recombinant t cells expand dramatically in patient blood, thus relative weight of other subtype t-cells in the total population decreases accordingly.figure v-j pairing events identified from p-vdj tagged reads. a), circos maps illustrating pairing frequencies of v-segments and j-segments from vj-containing reads; b), chow-ruskey diagram showing the five-way overlap of v-j pairing events in 010_m, 221_m, 347_b, 392_b with normal pool. characterization of “immune signature” – cdr3 in immune system, specific t cell clone can expand dramatically in response to tumor antigens. counting analysis of v-j pairing showed less number of pairing in malignant meningiomas compared to benign meningiomas or normal controls. but when we counted v-j pairing at a cutoff of 5% of total population, we found more pairing events in malignant than benign or normal.we then asked whether significantly expanded v-j pairing events in malignant patients could result from clonal expansion of particular t cells in which the sequence of cdr region can serve as a signature. we chose the v-j pairing events that are greatly expanded in either malignant individual compared to normal controls for cdr sequence analysis. in total, eight pairing events trbv6- with trbj1- trbv with trbj1- trbv3- with trbj2- trbv24- with trbj1- trbv with trbj2- trbv29- with trbj1- trbv7- with trbj1- and trbv4- with trbj2- were identified as dramatically increased in either one of the malignant samples. pairing of trbv7- and trbj that showed moderate increase in both malignant samples was also included for cdr analysis. except for pairing of trbv3- with trbj2- which has unusual cdr skipping, sequencing reads for every other combination showed one dominant fr3-cdr junction dna sequence, which accounts for 87% to 95% of the corresponding pairing event, suggesting clonal expansion. we also generated protein sequence logos for further comparison. in protein sequence logo plots, the amino acid sequences between the conserved cys at the 3’end of fr region and the common vfppey motif at the 5’end of trb constant region varied a lot for different v-j pairing events. in malignant meningiomas, both trbc and trbc subtypes were used in those expanded pairing events. as expected, most of the j subtypes fused to the closet known trbc subtypes. however, the dominant clone for trbv24- with trbj1- produced an unusual recombination/mutation at the beginning part of trb constant region that is not mappable to either trbc or trbc for pairing of trbv7- with trbj2- that showed expansion in both malignant samples, we observed that 88% to 90% of this population was from single dominant cdr signature. however, their translated cdr sequences were not identical, suggesting occurrence of other genomic editing events, such as hypermutation. in summary, cdr sequence logo analysis identified cdr signature sequences associated with individual malignant patient, which may reflect expansion of several specific v-j pairing clones in patient blood.figure sequence logos for detected fr3- trbc portions of malignant meningiomas. visualized in the dna sequence logos are the dominant clonal cdr sequences of selected v-j pairings; the translated protein sequence logos illustrate antigen recognition regions from the end of fr and the beginning of trbc. trbc and trbc sequences are underlined in purple and cyan colors, respectively. discussion and  in the current study, we presented an integrated approach by using single primer pcr together with next-generation sequencing to interrogate immune repertoire of tcr-beta. we have demonstrated the technical feasibility to use this system to infer immune repertoire, using whole blood from four meningiomas patients and two healthy donors. by aligning reads to a sequence database of germline v-genes, d-genes and j-genes, the usage of different v-gene segments was quantified. interestingly, comparison between malignant, benign and normal groups identified an increased usage of trbv trbv6- and trbv7- in malignant meningiomas. however, the pairing of v-j subtypes for recombination revealed a generally diversified immune repertoire for individual patient, although trbv7- with trbj appears to be associated with malignant transformation. further analysis of cdr region sequence logos of the top expanded v-j pairing in malignant meningiomas indicated distinct cdr signatures for the two malignant patients. however, we caution that these observations were made on a small number of samples, and they may not have any biological significance. our purpose is to use these data to demonstrate the technical feasibility of “single-primer” interrogation of immune repertoire, rather than determining what differs between malignant and benign tumors. there are several unique aspects of our protocol, compared to previous studies. first of all, total rna is extracted directly from frozen blood samples for profiling, thus the procedure can be easily adapted for clinical application. second, by using ligation-anchored pcr for amplification, all the recombination events at a particular immune gene locus is likely to be amplified in an unbiased manner. furthermore, sequencing of barcoded libraries through illumina hi-seq ensures fast turn-around time and good sequencing depth at a relatively low cost. finally, we recognize that more recent generations of illumina sequencers can now sequence bp or even continuous bp reads, potentially further reduce the computational complexity and increase the rate of recovering full length vj recombination for our approach. there are several major limitations of our protocol as well. first, due to the need to add in 3’-adaptors to the cdna terminus for ligation-anchored pcr, our method relies on rna samples, which are less readily available and more vulnerable to degradation, compared to genomic dna samples. however, in the current study, we used frozen whole blood samples and still obtained satisfactory results, suggesting that it is practically feasible to use this method in real-world clinical settings. second, although illumina hi-seq provided longer read length than illumina hi-seq to cover immune signature region cdr longer read length is still needed to cover the entire variable region of immune genes. thus the latest illumina miseq with bp or 2x bp chemistry should be more suitable for profiling immune repertoire. third, our sequencing data showed higher sequencing depth of malignant sample libraries compared to benign ones. we acknowledge that uneven mixing of the constructed libraries for sequencing could be the reason. to address the issue of sequencing depth, we performed additional data analysis using the first million sequencing reads of 010_m and 221_m data. of note, the results from the 7-million-read data were consistent with the conclusions from the whole sequencing data, indicating that the sequencing depth in our study is deep enough. fourth, we specifically designed primers to target tcr-beta isoforms. our sequencing data contained some other tcr isoforms as well, suggesting the presence of some minor cross reactions. our future studies may design specific primers for other types of isoforms as well, to investigate whether it is feasible to interrogate all major tcr isoforms in the same sequencing run. finally, the sample size in our study is small, which prevents us to draw any statistically significant conclusions on clonal expansion of trb subtypes associated with malignant transformation of meningiomas; however, the results from high-throughput sequencing data analysis do support validity of the developed integrated approach. in conclusion, we have developed a ligation-anchored pcr approach to interrogate immune repertoire, and demonstrated its technical feasibility and effectiveness in capturing the tcr-beta landscapes. recently, human antibody repertoire has been reported to change in response to influenza vaccination. we expect further development of our technology may enable a comprehensive delineation of immune repertoire, including other forms of tcrs as well as immunoglobulins. 
an increased usage of specific v segments and v-j pairing were observed in malignant meningiomas samples. multiplex pcr has been previously developed to assay immune repertoire; however, the use of primer pools leads to inherent biases in target amplification. further development of this technology may enable a comprehensive delineation of immune repertoire, including other forms of tcrs as well as immunoglobulins. next-generation sequencingimmune repertoirebloodissue-copyright-statement© the author 2015 mapping of sequencing data showed that 64% to 96% of mapped tcrv-containing reads belong to trb subtype. by utilizing a universal primer paired with a single primer targeting the conserved constant region, we amplified tcr-beta variable regions from total rna extracted from blood. as one of the genetic mechanisms for adaptive immunity, vj recombination generates an enormous repertoire of t-cell receptors. with the development of high-throughput sequencing techniques, systematic exploration of vj recombination becomes possible. next-generation sequencing libraries were then prepared for illumina hiseq sequencer, which generates 151-bp read length to cover the entire vj recombination region. we demonstrated the technical feasibility and effectiveness of ligation-anchored pcr approach in capturing the tcr-beta landscapes. we evaluated this approach on blood samples from healthy donors and from patients with malignant and benign meningiomas.
 as one of the genetic mechanisms for adaptive immunity, vj recombination generates an enormous repertoire of t-cell receptors. with the development of high-throughput sequencing techniques, systematic exploration of vj recombination becomes possible. multiplex pcr has been previously developed to assay immune repertoire; however, the use of primer pools leads to inherent biases in target amplification. in our study, we developed a “single-primer" ligation-anchored pcr method that may amplify the repertoire with much less biases.  by utilizing a universal primer paired with a single primer targeting the conserved constant region, we amplified tcr-beta variable regions from total rna extracted from blood. next-generation sequencing libraries were then prepared for illumina hiseq sequencer, which generates 151-bp read length to cover the entire vj recombination region. we evaluated this approach on blood samples from healthy donors and from patients with malignant and benign meningiomas. mapping of sequencing data showed that 64% to 96% of mapped tcrv-containing reads belong to trb subtype. an increased usage of specific v segments and v-j pairing were observed in malignant meningiomas samples. the cdr sequences of the expanded v-j pairs were distinct in each malignant individual, even for pairing of trbv7- with trbj2- that showed increased usage in both cases.  we demonstrated the technical feasibility and effectiveness of ligation-anchored pcr approach in capturing the tcr-beta landscapes. further development of this technology may enable a comprehensive delineation of immune repertoire, including other forms of tcrs as well as immunoglobulins. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords next-generation sequencingimmune repertoirebloodissue-copyright-statement© the author 2015 