Patent Application
20220220470
Single cell transcriptional analysis reveals a wealth of information that is obscured when analyzing the RNA of a sample en masse. Furthermore, it is important to know the identity of the T Cell Receptor (TCR) or B Cell Receptor (BCR) chain VDJ-rearrangements associated with the phenotypic state of each individual cell. A TCR gene consists of numerous V regions (variable region, V), J regions (joining region, J), D regions (diversity region, D), and constant regions, C regions (C) encoded by different regions in the genome. In T cell differentiation process, such gene fragments are genetically rearranged in various combinations. This information is valuable as it can provide direct calculations of clonal frequency in various cell subsets, tracking of specific lymphocytes with treatment, and reveal paired information for both chains of the receptor for downstream functional analysis or Car-T development. Many single cell systems on the market utilize an oligo-dT-based reverse transcription (RT) step in which the RNA from an isolated single cell is reverse transcribed using a barcoded oligo-dT primer. The resulting RNA sequencing can provide information related to various genes expressed in the cell traced back to the same cell via the barcode introduced during RT, but this method cannot reliably reveal the sequence of the paired-receptor VDJ-rearrangement.
This deficiency is due, in part, to the fact that the de novo VDJ-rearrangement is far from the poly-A tail of the RNA making sequencing on certain high throughput next-generation sequencing platforms a challenge, because the resulting products are 1-1.5 kb in length. This can be overcome by switching to longer read length NGS platforms or using commercially available Illumina sequencing kits in a unique manner. This switch, however, still does not overcome the deficiencies of conventional methods.
In some embodiments, the present disclosure relates to a method comprising the steps of: reverse transcribing at least one first strand of cDNA from mRNA using an oligo-dT primer, wherein the mRNA comprises at least one target sequence to produce a first strand cDNA and wherein the oligo-dT primer comprises an engineered sequence on the 5′ end; producing a first set of amplicons by amplifying the first strand cDNA using a multiplex primer mix, wherein the multiplex primer mix comprises two or more primers configured to bind two or more sequences selected from the group consisting of: TCR alpha chain VDJ and TCR beta chain VDJ sequences, and a reverse primer configured to bind to the engineered sequence on the 5′ end of the oligo-dT primer; using probe beads to capture amplicons comprising TCR alpha chain VDJ sequences and TCR beta chain VDJ sequences from the first set of amplicons, wherein the probe beads comprise primers configured to bind to the TCR alpha chain constant gene region and the TCR beta chain constant gene region; washing the sample to remove uncaptured amplicons from the first set of amplicons; eluting captured amplicons from the probe beads to produce a pool of eluted amplicons; and amplifying the eluted amplicons using PCR to produce a second set of amplicons.
In some embodiments, the method further comprises the step of, after amplifying the eluted amplicons, sequencing the second set of amplicons using next generation sequencing. In some embodiments, the method further comprises the step of, after sequencing the second set of amplicons using next generation sequencing, evaluating the sequences to determine the frequency of TCR alpha chain VDJ sequences and TCR beta chain VDJ sequences.
In some embodiments of the method, the probe bead primers are configured to bind to at least one location within the TCR alpha chain constant gene region and at least one location within the TCR beta chain constant gene region. In some embodiments of the method, the probe bead primers are configured to bind to at least one location within the TCR alpha chain constant gene region. In some embodiments of the method, the probe bead primers are configured to bind to at least one location within the TCR beta chain constant gene region.
In some embodiments of the method, the oligo-dT primer comprises a molecular barcode. In some embodiments of the method, the multiplex primer mix comprises an engineered sequence on the 5′ end of the primers and which is configured as a universal binding site.
In some embodiments of the method, a majority of the two or more primers of the multiplex primer mix are configured to favor amplification of the sense strand VDJ sequences. In some embodiments of the method, a majority of the two or more primers of the multiplex primer mix are configured to bind to one or more TCR alpha chain VDJ sequences. In some embodiments of the method, a majority of the two or more primers of the multiplex primer mix are configured to bind to one or more TCR beta chain VDJ sequences.
In some embodiments of the method, prior to using probe beads, the first set of amplicons is cleaned by SPRI bead selection and an additional round of PCR is performed.
The disclosure can be better understood with reference to the following drawings. The elements of the drawings are not necessarily to scale relative to each other, emphasis instead being placed upon clearly illustrating the principles of the disclosure. Furthermore, like reference numerals designate corresponding parts throughout the several views.
This disclosure generally relates a method of amplifying TCR alpha chain VDJ and/or TCR beta chain VDJ sequences by reverse transcribing at least one first strand of cDNA from mRNA containing at least one target sequence using an oligo-dT primer, using a multiplex primer mix of primers configured to bind to TCR alpha chain VDJ and/or TCR beta chain VDJ sequences to amplify the cDNA, using probe capture to capture TCR VDJ-specific amplicons and elute non-TCR VDJ-specific amplicons, performing an additional round of PCR to further enrich TCR alpha chain VDJ and TCR beta chain VDJ amplicons, and sequencing the resulting amplicons using next generation sequencing.
As used herein, the term “engineered sequence” refers to a nucleotide sequence. An engineered sequence can be configured for a particular purpose. For example, an engineered sequence can be designed to serve as a universal binding site.
As used herein, the term “oligo-dT” refers to a homopolymer typically consisting exclusively of thymidines. Preferred length of the oligo-dT molecule is 10 to 100 bases, more preferred is a length of 10 to 30 bases, most preferred is a length of 20 bases. The oligo-dT molecule is preferably blocked at its 3′-hydroxyl group by a blocking group to prevent extension by a polymerase reaction. It is known to those of skill in the art that oligo-dT can also be modified as long as the molecule is capable of hybridizing to polyA-RNA. Modifications are but not limited to all types of thymidine analogs like 2′-deoxyuridine. 2′-O-methyl-uridine, LNA (locked nucleic acid) thymidine or uridine derivatives, PNA (peptide nucleic acid) thymine or uracil derivatives, HNA (hexitol nucleic acid) thymidine or uridine derivatives or base modified uracil derivatives like 5-propinyl-uracil, but also modification with labels like fluorescent labels or haptens or modification with nucleotides or nucleotide sequences other than thymidine and uridine.
As used herein, the term “probe bead” means a bead to which one or more primers can be adhered and which is suitable for use with one or more purification methods, such as column purification.
As used herein, the term “subject” means a human or other animal. In some embodiments, the subject has been immunized or is suffering from an infection, cancer, an autoimmune condition, or any other diseases. For example, the subject can be a human diagnosed with a disease, exhibiting symptoms of a disease, not diagnosed with a disease, or not exhibiting symptoms of a disease.
As used herein, the term “target sequence” means a nucleic acid sequence to be detected and which anneals with a probe or primer under hybridization, annealing or amplification conditions.
As used herein, the term “TCR alpha chain” and “TCR beta chain” refer to the alpha and beta chains that comprise the TCR and which assemble to form a heterodimer and associate with the CD3-transducing subunits to form the T-cell receptor complex present on the T cell surface. Each alpha and beta chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. As for immunoglobulin molecules, the variable region of the alpha and beta chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T cells.
In certain embodiments, this disclosure relates to a method comprising the steps of: reverse transcribing at least one first strand of cDNA from mRNA, wherein the mRNA comprises at least one TCR alpha chain VDJ or TCR beta chain VDJ sequence, using an oligo-dT primer. In some embodiments, the oligo-dT primer comprises an engineered sequence 5′ of the oligo-dT portion. The engineered sequence 5′ of the oligo-dT portion provides the benefit of simplifying downstream amplification. In others embodiments, the oligo-dT primer does not comprise an engineered sequence 5′ of the oligo-dT portion. The oligo-dT primer may also comprise a molecular barcode as a unique identifier. For example, a molecular barcode can comprise a nucleotide sequence comprising from about 4 to 15 randomly-generated nucleotides. Molecular barcodes can be used as disclosed in US20150132754 (Wang, et al.) and US20120171725 (Han), which are incorporated herein by reference.
The mRNA can be isolated from any source comprising one or more T cells. For example, the mRNA can be isolated from peripheral blood or other biological samples from a subject. Methods of extracting mRNA are known to those of skill in the art.
The first strand cDNA produced after reverse transcription with the oligo-dT primer is then amplified using a targeted multiplex mix of primers covering either TCR alpha chain VDJ only or TCR beta chain VDJ only, or both TCR alpha chain and beta chain VDJ. The VDJ-primer mixes may also contain an engineered sequence on the 5′ end to provide a universal binding site for downstream PCR. The multiplex PCR mix comprises an optimized primer set of VDJ sequences of interest and a reverse primer for the engineered sequence located on the 5′ end of the oligo-dT primer during reverse transcription (
In certain embodiments, PCR is performed using an asymmetric balance of primers configured to favor production of the sense strand VDJ. In certain embodiments, the PCR product of the first amplification is cleaned by either SPRI bead selection or another standard PCR clean-up method, and the PCR is repeated to increase the template amount. The applicants discovered that even if the library was sequenced after the completion of the one or more asymmetric PCR steps, the alpha chain VDJ sequences were still very infrequent in the sequencing data with respect to the beta chain sequences.
Next, the entire PCR product is selected via probe capture. Briefly, probe beads are generated using constant region capture primers specific for the TCR alpha chain constant gene region and the TCR beta chain constant gene region. These capture sequences can be for one specific location or multiple locations within the junction or constant gene for their respective chains. The location of the junction or constant probe primer is also an important consideration as some positions are better than others for VDJ sequence recovery. After the hybridization step, unbound species are washed away—for example by using column purification methods known to those of skill in the art—and the captured sequences are eluted from the beads. Another round of PCR is then performed to both enrich the template again and assign additional indices for sequencing. The library is then sequenced using next generation sequencing, and the data are evaluated for frequency of TCR alpha chain and beta chain VDJ sequences.
During the development of the presently disclosed methods to amplify the VDJ-rearrangements using a multiplex mix of primers covering the TCR beta chain and the TCR alpha chain (a targeted-sequencing approach), the applicants discovered that the VDJ information for the alpha chain was consistently very difficult to capture from an oligo-dT reverse transcribed cDNA, regardless of whether the system was a single cell capture system or a bulk RNA reverse-transcribed by an oligo-dT primer. Since this information forms one of the chains of receptor pair, it is critical that this information also be recovered.
An advantage of the disclosed methods is to consistently recover both the TCR alpha and beta chain VDJ-rearrangement information using a targeted-sequencing approach coupled to probe-capture. These methods rescue the sequence of the TCR alpha chain VDJ, while also increasing the number of VDJ-specific sequencing reads. As a result, the presently disclosed methods result in a much higher capture of alpha chain sequences relative to conventional methods, thereby increasing the discovery of the alpha chain immune repertoire and the overall receptor pairing success rate. The methods also enable an increased discovery of TCR beta chain sequences. Further, the reduction in the number of off-target sequencing reads offered by the disclosed methods results in overall reduced experiment associated costs, because less sequencing depth is required to maximize the clonotype discovery for each chain. As a result, both immune repertoire discovery and single cell pairing from an oligo-dT reverse transcribed template is improved by the described methods.
The TCR alpha chain VDJ, the TCR beta chain VDJ, and both together were amplified on reverse transcribed cDNA on bulk RNA. Analysis of the NGS data revealed approximately 90-94% of the data was unrelated to the TCR VDJ sequences, approximately 8% of TCR beta chain sequences and approximately 6% of TCR alpha chain sequences. When the TCR alpha and beta chain sequences were co-amplified, approximately 92% of the data were unrelated to the VDJ sequences, with 5.8% TCR beta chain sequences, and 1.9% TCR alpha chain sequences.
When this experiment was repeated on reverse-transcribed RNA from a single cell system, a similar result was achieved with 89-95% of the data unrelated to the TCR VDJ sequences, 11% of TCR beta chain sequences and 5% of TCR alpha chain sequences. All data unrelated to the VDJ information was considered “trash” since applicants expected, in a targeted sequencing assay, for more than 98% of the data to contain rearranged VDJ sequences. This is the case when a constant region gene primer and a V-primer mix is used during the amplification. This “trash” data results in wasted sequencing reads and dropout of VDJ-rearrangements of interest, thus increasing experiment costs and reducing the VDJ-paired receptor success rates.
After analysis of the NGS data, applicants discovered that much of the trash was a result of one primer, V-alpha 4. Therefore, applicants repeated the experiment using the original primer mix and a primer mix in which V-alpha 4 primer was removed. Applicants also tested multiple ways to wash the beads to help remove any unwanted “trash”. Again, analysis of the NGS data revealed only a slight 2% improvement of TCR alpha chain discovery with 92% of the data unrelated to the TCR VDJ sequences (trash) and 8% of TCR alpha chain sequences. Applicants then attempted a different primer set where the V-primers were in a completely different position (closer to the 5′ end of the RNA). Again, the same issue persisted regardless of the primer position. Applicants also attempted a method in which the long read primer set was utilized in the first round of PCR, followed by additional PCR with the short read V-primer mix. The idea here being that using two primer sets specific for the V-alpha region would cumulatively result in an increase of V-alpha sequences. This adjustment did not improve the results either.
The off-target sequencing results were re-analyzed for the percentages of various components to see if certain V-alpha sequences could be modified or removed to increase the percentage of on-target V-alpha results. Using all of the information related to the off-target amplicons, the entire primer system was redesigned to avoid off-target results. The analysis on applicant's bulk RNA oligo-dT system was repeated. Analysis of the NGS data reveals 90% of the data were unrelated to the TCR VDJ sequences (trash) and 6-7% of TCR alpha chain sequences. Thus, even redesigning the primers did not solve the issue of off-target amplification when amplifying the TCR alpha chain VDJ specifically, demonstrating that the TCR alpha chain locus is difficult to amplify with gene-specific primers from cDNA generated with an oligo-dT primer. This difficulty persisted even after modifying the PCR strategy, modifying various PCR clean-up steps, and redesigning the gene-specific primers used for amplification.
To ascertain whether the issues with the TCR alpha chain were due to inefficient reverse transcription of the TCR alpha chain, applicants tested the first strand cDNA by amplifying with a V- and C-specific primer mix to make sure that the oligo-dT conversion of TCR alpha strand RNA was occurring and to assess the number of TCR alpha chain VDJ sequences that were actually generated as first strand cDNA products. For the same library without performing this enrichment step, applicants discovered approximately 300 clonotypes. However, after enrichment applicants discovered approximately 1,600 clonotypes. These data demonstrate that the TCR alpha chain first strand cDNA is present. Thus, the dropout in TCR alpha chain is happening at a later stage of PCR (not during RT) due to competition from off-target species in the oligo-dT mix.
For Bulk RNA, NGS data reveals by using no probe pull down for TCR alpha chain only, 85% of the data were unrelated to the TCR VDJ (trash). TCR beta chain only reveals 87% of the data were unrelated to the TCR VDJ. When co-amplifying both TCR alpha and beta chain sequences using no probe to capture, 73% of the data were unrelated to the TCR VDJ sequences, 24% of TCR beta chain sequences and 2% of TCR alpha chain sequences (
When this was repeated in the context of a single cell system and the absence of probe-capture with TCR alpha chain, 85% of the data were unrelated to the TCR VDJ sequences (trash). TCR beta chain only reveals 83% of the data were unrelated to the TCR VDJ sequences. When co-amplifying both TCR alpha and beta chain sequences using the non-probe capture system, 85% of the data were unrelated to the TCR VDJ sequences, 11% of TCR beta chain sequences and 3% of TCR alpha chain sequences (
The systems, methodologies and the various embodiments thereof described herein are exemplary. Various other embodiments of the systems and methodologies described herein are possible.
This application claims priority to International Application No. PCT/US2020/046433 entitled “Probe-Capture Method for TCR Alpha and Beta Chain VDF-Recovery from Oligo-DT Reverse Transcribed RNA” and having an international filing date of on Aug. 14, 2020 and U.S. Provisional Patent Application No. 62/886,663, entitled “Probe-Capture Method for TCR Alpha and Beta Chain VDF-Recovery from Oligo-DT Reverse Transcribed RNA” and filed on Aug. 14, 2019, which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/046433 | 8/14/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62886663 | Aug 2019 | US |
