Patent Application
20210317522
The present application claims priority from Australian Provisional Application No. 2018903546 filed on 21 Sep. 2018, the full contents of which is incorporated herein by reference.
The present disclosure relates to an improved methodology for phenotyping and molecular characterisation of single cells using high-throughput and multiplexed targeted long-read single cell sequencing. In one particular example, the present disclosure relates to a methodology which combines targeted long-read sequencing with short-read based transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning for high throughput deep single cell profiling.
B and T lymphocytes recognise foreign and self-antigens through their antigen receptors which in turn govern their development, survival and activation. To establish a diverse repertoire of antigen-specific lymphocytes, the T Cell Receptor (TCR) and B Cell Receptor (BCR) are assembled from variable (V), diversity (D) and joining (J) gene segments in a somatic process known as V(D)J recombination (Bassing et al., (2002) Cell, 109(Suppl):545-55). Random addition or removal of nucleotides at the complementarity determining region 3 (CDR3), which adjoins V(D)J junctions, largely determines the specificity towards antigen. Due to the significant diversity of both the BCR and TCR repertoire, estimated at >1012 (Calis and Rosenberg (2014) Trends in immunology, 35(12):581-90; Laydon et al., (2015) Biological sciences, 370(1675)), it is highly likely that two cells carrying the same antigen receptor sequence are clonally related and constitute a clonotype. As a result, when a B cell or T cell clone undergoes clonal expansion the identity of a BCR or TCR sequence serves as a unique clonal identifier or ‘clonal barcode’ and provides information on antigen specificity and clonal ancestry.
Sequencing the BCR or TCR of individual lymphocytes in parallel with their transcriptome provides high resolution insights into the adaptive immune response in a range of disease settings such as infectious disease, autoimmune disorders and cancer. A common approach to link paired antigen receptor sequences with gene expression profiles of single lymphocytes is through the use of the full-length scRNA-seq method SmartSeq2 (Picelli et al., (2013). Nature methods, 10(11):1096-8), where computational methods can reconstruct paired TCRαβ sequences or paired IgH and IgL sequences from Illumina short-reads (Afik et al., (2017) Nucleic acids research, 45(16):e148; Eltahla et al., (2016) Immunology and cell biology, 94(6):604-11; Stubbington et al., (2016) Nature methods, 13(4):329-32; Upadhyay et al., (2018) Genome medicine, 10(1):20). However, SmartSeq2 generally relies on plate- or well-based microfluidics and is therefore limited in the number of cells that can be processed, typically 10s to 100s. Additionally, a large number of sequencing reads are generally required to computationally reconstruct paired antigen receptors. As such, the cost per cell is relatively high ($50-$100 USD).
Recent technological advancements in high-throughput scRNA-seq methods allow thousands of cells to be captured and sequenced in a relatively short time frame and at a fraction of the cost (Ziegenhain et al., (2017) Molecular cell., 65(4):631-43.e4). Such methods rely on capture of polyadenylated (polyA) mRNA transcripts followed by cDNA synthesis, pooling, amplification, library construction and Illumina 3′ or 5′ cDNA sequencing. The combination of fragmentation and short-read sequencing fails to sufficiently sequence the V(D)J regions of rearranged TCR and BCR transcripts, which are located closer to the 5′ end of the transcript. Consequently, 3′-tag scRNA-seq platforms have limited application for determining clonotypic information from large numbers of lymphocytes. Recent advances in long read sequencing technologies present a potential solution to the shortcomings of short-read sequencing. Full-length cDNA reads can encompass the entire sequence of BCR and TCR transcripts, but typically suffer from higher error rates and lower sequencing depth than short read technologies.
Accordingly, there is a need for improved methodologies for high-throughput and multiplexed targeted long-read single cell sequencing, such as for use in cellular phenotyping.
The present disclosure is based, at least in part, on the recognition by the inventors that, although existing high-throughput single-cell RNA-seq (scRNA-seq) is a powerful tool for gene expression profiling of complex and heterogeneous biological systems, e.g., such as the immune system or populations of clonally diverse cancer cells, these existing methods only provide short-read sequence data from one end of a cDNA template, which is poorly suited to the investigation of gene-regulatory events such as alternative transcript isoforms or fusion genes, adaptive immune responses or somatic genome evolution. The inventors have therefore developed a method that combines targeted long-read sequencing with short-read based transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning. The present inventors have then used this methodology, termed Repertoire And Gene Expression by sequencing (RAGEseq), to accurately characterise T-cell (TCR) and B-cell (BCR) receptor transcripts and transcriptional profiles of more than 7138 lymphocytes sampled from the primary tumour and draining lymph node of a breast cancer patient. In doing so, the inventors were able to phenotype clonally-related lymphocytes between tissues, identify alternately-spliced BCR transcripts encoding receptors destined for secretion versus membrane localization, and reveal somatic hypermutation of BCRs. The inventors also used this methodology to analyse PTPRC splice variants encoding alternate isoforms of CD45, providing important information on whether lymphocytes are naive (CD45RA) or antigen-experienced (CD45RO). In addition to the valuable insight this may provide to an immunologist, it demonstrates the use of RAGE-seq to analyse splicing of a transcript which is less abundant than TCRs/BCRs. These results demonstrate that RAGE-seq is an accessible and cost-effective method for high throughput deep single cell profiling, applicable to a wide diversity of biological questions and challenges. These include tumour immunology, autoimmune disease, somatic mutation and clonal evolution in cancer and adaptive resistance to therapy.
Accordingly, in one example, the disclosure provides a method for high-throughput and multiplexed phenotyping and characterisation of single cells, said method comprising:
In one example, unique tissue barcodes are assigned and introduced to the nucleic acid molecules at step (a) to enable subsequent pooling and multiplex analysis of nucleic acid molecules derived from more than one tissue type and/or sample e.g., including samples from different subjects.
The library of nucleic acid molecules may contain any nucleic acid molecule selected from the group consisting of cDNA, genomic DNA, barcodes, cellular RNA (e.g., such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA) and/or non-coding RNA (ncRNA)) and combinations thereof. In one example, the library of nucleic acid molecules comprises cDNA. In one example, the library of nucleic acid molecules comprises genomic DNA. In one example, the library of nucleic acid molecules comprises barcodes. In one example, the library of nucleic acid molecules comprises cellular RNA e.g., such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA) and/or non-coding RNA (ncRNA). In one example, the library of nucleic acid molecules comprises a mixture of cDNA, genomic DNA, barcodes and cellular RNA.
In on example, the method further comprises a single cell capture step prior to step (a). For example, single cell capture may be performed by one or more of the following means: a droplet-based microfluidics platform, a flow cytometry platform, a plate-based platform, a microwell-based platform or any combination thereof.
In one example, the method described herein further comprises a step of isolating the cells prior to the single cell capture step e.g., by disassociating tissue or bodily fluid into cellular components or by selection of one or more subsets of cells from said tissue or bodily fluid. The first library component may be sequenced using a short-read sequencing method and/or a long-read sequencing method. In one example, first library component is sequenced using a short-read sequencing method. In one example, the first library component is sequenced using a long-read sequencing method. In yet another example, the first library component is sequenced using both short and long-read sequencing methods.
In one example, the short-read sequencing method is a next generation sequencing (NGS) method selected from the group consisting of sequencing-by-hybridization, sequencing-by-synthesis, sequencing-by-ligation platform, ion semiconductor sequencing, combinatorial probe anchor synthesis, and combinations thereof. In one example, short-read sequencing may be performed using a sequencing-by-hybridization method. In one example, short-read sequencing may be performed using a sequencing-by-synthesis method. In one example, short-read sequencing may be performed using a sequencing-by-ligation method. In one example, short-read sequencing may be performed using ion semiconductor sequencing. In one example, short-read sequencing may be performed using a combinatorial probe anchor synthesis method. In another example, short-read sequencing may be performed using a combination of sequencing-by-hybridization, sequencing-by-synthesis, sequencing-by-ligation, ion semiconductor sequencing and combinatorial probe anchor synthesis methods.
In one example, the long-read sequencing method is a nanopore sequencing method, a single molecule real time (SMRT) sequencing method or a combination thereof. In one example, the long-read sequencing method is a nanopore sequencing method. In another example, the long-read sequencing method is a SMRT sequencing method. In yet another example, a combination of nanopore sequencing and SMRT sequencing is used.
In one example, the method comprises targeted enrichment of the first and/or second library components for sequences or features of interest prior to sequencing. In one example, the method comprises targeted enrichment of the second library component only. In another example, the method comprises targeted enrichment of the first and second library components. Targeted enrichment prior to sequencing may be performed using a hybridisation capture protocol. One exemplary hybridisation capture protocol relies on biotinylated hybridisation beads attached to capture probes which bind selectively to genetic, epigenetic or transcriptomic sequences or features or interest within the library component(s). However, other hybridisation capture method known in the art may also be used. Alternatively or in addition to the use of a hybridisation capture protocol, targeted enrichment of the library component(s) may comprise depleting unwanted sequences or features from the library component(s) prior to sequencing.
Alternatively, or in addition, the targeted enrichment may be performed in silico post-sequencing e.g., computational enrichment of sequence data based using filters. In accordance with this example, the in silico enrichment may preferentially select for sequences or features of interest in the first and/or second sets of sequence data. In another example, the in silico enrichment may deplete unwanted sequences or features from the first and/or second sets of sequence data.
In any one of the examples, targeted enrichment increases representation of sequences or features of interest with the first and/or second set of sequence data, particularly within the second set of sequence data. In accordance with any of the examples relating to targeted enrichment, the second set of sequence data may be enriched for T and/or B cell receptor sequences. However, a skilled person will appreciate that the sequence data may be enriched for any gene(s), sequence(s) and/or feature(s) of interest. For example, the sequence data may be enriched for immunological genes e.g., PTPRC encoding CD45. In one example, the molecular characterisation of the contigs is on the basis of one or more of the following: antigen receptor clonotyping, mutation analysis, somatic genome variation, alternative transcript splicing, fusion genes or chimeric transcripts, transcript isoform quantification and combinations thereof. However, depending on the enrichment performed (if any), the starting cell population(s) and/or the sequences of interest, the molecular characterisation may be based on other features of interest.
In accordance with one example in which targeted enrichment was performed for T and/or B cell receptor sequences, either before or after sequencing as described herein, molecular characterisation of the contigs may be performed using IgBlast.
Alternatively or in addition, the molecular characterisation of the contigs is on the basis of one or more of the following:
In one example, the method described herein comprises performing one or more filtering steps on the second set of sequence data to remove sequences which are below a desired length (e.g., <500 bases long), uninformative, erroneous and/or not of interest. Filtering may also involve removing adapter sequences added to sequences during preparation of the nucleic acid library. The filtering step may be performed on the second set of sequence data comprising long-read sequences at any time prior to de novo assembly into contigs. For example, the one or more filtering steps may be performed prior to demultiplexing the second set of sequence data. Alternatively, or in addition, one or more filtering steps may be performed after the demultiplexing step but prior to de novo assembly of long-read sequences into contigs.
In one example, demultiplexing of the second set of sequence data is supervised. For example, supervised demultiplexing may comprise comparing or matching the long-read sequences to the corresponding sequences in the first set of sequence data using the UMI, unique cell barcodes, optionally unique tissue barcodes or combinations thereof.
In another example, demultiplexing of the second set of sequence data is unsupervised. For example, unsupervised demultiplexing may comprise comparing the second set of sequence data to itself in a manner sufficient to identify commonalities in long-read sequences, or identifying sequence features selected from UMI, unique cell barcodes and/or unique tissue barcodes.
In yet another example, the demultiplexing of the second set of sequence data involves both supervised and unsupervised methods.
Assignment of the demultiplexed long-read sequences into one or more groups may comprise de novo assembly of long-read sequences, alignment to one or more reference sequences, multiple sequence alignments or other approach capable of grouping the long-read sequences e.g., based on tissue type, cell type and/or sequence type. Grouped long-read sequences may then be assembled and a contig formed on the basis of consensus sequence. In one example, de novo grouping and assembly of the demultiplexed long-read sequences into one or more contigs is performed using CANU software. In another example, grouping and assembly of the demultiplexed long-read sequences is performed by aligning the long sequence reads to sequences of interest corresponding to the enrichment targets of step (d) using the Minimap2 software, followed by multiple sequence alignment of aligned sequences using MAFFT software.
The method described herein may also comprise one or more consensus correction and/or polishing steps to correct errors in the long-read sequences and/or contigs and thereby improve the consensus sequence. The one or more correction and/or polishing steps may be performed after demultiplexing but before assembly into contigs. Alternatively, or in addition, the one or more correction and/or polishing steps may be performed on the contigs. For example, contig consensus correction may be performed using Minimap2 and/or RACON. For example, consensus polishing may be performed using Minimap2 and/or Nanopolish.
In some example, the method of the disclosure (RAGE-seq) may be performed in combination with one or more other analytical methods e.g., such as Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq). Such a combinatorial multi-omic methodology may permit the simultaneous phenotyping of cellular populations using e.g., protein targets plus RNA with full length sequencing capacity. In accordance with an example in which the method of the disclosure is combined with CITE-seq, step (b) described hereinabove may comprise dividing the library into at least three components comprising the first library component, the second library component and a third library component, wherein the third library component comprises CITE-seq barcodes from antibodies.
The disclosure also provides a computer implemented method for phenotyping and characterising single cells using data obtained from high-throughput and multiplexed long-read single cell sequencing, said method comprising:
(d) demultiplexing the second set of sequence data to distinguish between individual long-read sequences;
In one example, each nucleic acid molecule comprises a unique tissue barcode to enable pooling and deconvolution of sequence data for nucleic acid molecules derived from more than one tissue type and/or sample e.g., including samples from different subjects.
The library of nucleic acid molecules may contain any nucleic acid molecule selected from the group consisting of cDNA, genomic DNA, barcodes, cellular RNA (e.g., such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA) and/or non-coding RNA (ncRNA)) and combinations thereof. In one example, the library of nucleic acid molecules comprises cDNA. In one example, the library of nucleic acid molecules comprises genomic DNA. In one example, the library of nucleic acid molecules comprises barcodes. In one example, the library of nucleic acid molecules comprises cellular RNA e.g., such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA) and/or non-coding RNA (ncRNA). In one example, the library of nucleic acid molecules comprises a mixture of cDNA, genomic DNA, barcodes and cellular RNA.
The first set of sequence data may be generated by a short-read sequencing method and/or a long-read sequencing method. In one example, the first set of sequence data has been generated using a short-read sequencing method. In one example, the first set of sequence data has been using a long-read sequencing method. In yet another example, the first set of sequence data has been using both short and long-read sequencing methods.
In one example, the short-read sequencing method is a next generation sequencing (NGS) method selected from the group consisting of sequencing-by-hybridization, sequencing-by-synthesis, sequencing-by-ligation platform, combinatorial probe anchor synthesis and combinations thereof. In one example, short-read sequencing method is a sequencing-by-hybridization method. In one example, the short-read sequencing method is a sequencing-by-synthesis method. In one example, short-read sequencing method is a sequencing-by-ligation method. In one example, short-read sequencing method is a combinatorial probe anchor synthesis method. In another example, short-read sequencing method is a combination of sequencing-by-hybridization, sequencing-by-synthesis, sequencing-by-ligation, ion semiconductor sequencing and combinatorial probe anchor synthesis methods.
In one example, the second set of sequence data received has been generated by a nanopore sequencing method or a single molecule real time (SMRT) sequencing method. In one example, the second set of sequence data received has been generated using a nanopore sequencing method. In another example, the second set of sequence data received has been generated using a SMRT sequencing method. In yet another example, the second set of sequence data has been generated using a combination of nanopore sequencing and SMRT sequencing.
In one example, the first and/or second set of sequence data is enriched for genetic, epigenetic or transcriptomic sequences or features or interest. In one example, the second set of sequence data is enriched for genetic, epigenetic or transcriptomic sequences or features or interest. In another example, the first and second sets of sequence data are both enriched for genetic, epigenetic or transcriptomic sequences or features or interest. In accordance with an example in which the first and/or second sets of sequence data is/are enriched, targeted enrichment may have occurred prior to sequencing, following sequencing (e.g., by in silico means) or both. In one example, target enrichment comprises actively selecting for sequences or features or interest e.g., using a hybridisation capture protocol prior to sequencing and/or by in silico means following sequencing. Alternatively or in addition, target enrichment may comprise depleting unwanted sequences or features prior to sequencing and/or by in silico means following sequencing. In accordance with any of the examples relating to targeted enrichment, the targeted enrichment may be for T and/or B cell receptor sequences. However, a skilled person will appreciate that the sequence data may be enriched for any gene(s) and/or feature(s) of interest. For example, in another example the sequence data may be enriched for immunological genes e.g., PTPRC encoding CD45.
In one example, molecular characterisation of the contigs is on the basis of one or more of the following: antigen receptor clonotyping, mutation analysis, somatic genome variation, alternative transcript splicing, fusion genes or chimeric transcripts, transcript isoform quantification and combinations thereof. However, characterisation of contigs may be based on other features depending on whether the second set of sequence data comprising long-read sequences has been enriched for any particular sequences or features of interest, the starting cell population(s) and/or the sequences of interest.
In accordance with an example in which targeted enrichment was performed for T and/or B cell receptor sequences, molecular characterisation of the contigs may be performed using IgBlast.
Alternatively or in addition, the molecular characterisation of the contigs may be on the basis of one or more of the following:
In one example, the computer implemented method described herein comprises performing one or more filtering steps on the second set of sequence data to remove sequences which are below a desired length (e.g., <500 bases long), uninformative, erroneous and/or not of interest. Filtering may also involve removing adapter sequences added to sequences during preparation of cDNA libraries. The one or more filtering step may be performed on the second set of sequence data at any time prior to de novo assembly into contigs. For example, the one or more filtering steps may be performed prior to demultiplexing the second set of sequence data. Alternatively, or in addition, one or more filtering may be performed after the demultiplexing step but prior to de novo assembly of long-read sequences into contigs.
In one example, demultiplexing of the second set of sequence data is supervised. For example, supervised demultiplexing may comprise comparing or matching the long-read sequences to the corresponding sequences in the first set of sequence data using the UMI, unique cell barcodes, unique tissue barcodes or combinations thereof.
In another example, demultiplexing of the second set sequence data is unsupervised. For example, unsupervised demultiplexing may comprise comparing the second set of sequence data to itself in a manner sufficient to identify commonalities in the long-read sequences, or identifying sequence features selected from UMI, unique cell barcodes and/or unique tissue barcodes.
In yet another example, the demultiplexing of the second set of sequence data involves both supervised and unsupervised methods.
Assignment of the demultiplexed long-read sequences into one or more groups may comprise de novo assembly of long-read sequences, alignment to one or more reference sequences, multiple sequence alignments or other approach capable of grouping the long-read sequences e.g., based on tissue type, cell type and/or sequence type. Grouped long-read sequences may be assembled and a contig formed on the basis of consensus sequence. In one example, de novo grouping and assembly of the demultiplexed long-read sequences into one or more contigs is performed using CANU software. In another example, grouping and assembly of the demultiplexed long-read sequences is performed by aligning the long reads to sequences of interest corresponding to the enrichment targets of step (d) using the Minimap2 software, followed by multiple sequence alignment of aligned sequences using MAFFT software.
The computer implemented method described herein may also comprise one or more consensus correction and/or polishing steps to correct errors in the long-read sequences and/or contigs and thereby improve the consensus sequence. The one or more correction and/or polishing steps may be performed after demultiplexing but before assembly into contigs. Alternatively, or in addition, the one or more correction and/or polishing steps may be performed on the contigs. For example, contig consensus correction may be performed using Minimap2 and/or RACON. For example, consensus polishing may be performed using Minimap2 and/or Nanopolish.
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, feature, composition of matter, group of steps or group of features or compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, features, compositions of matter, groups of steps or groups of features or compositions of matter.
Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.
Any example or embodiment of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant DNA, recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, is understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
As used herein, the term “unique molecular identifier”, “UMI” or similar refers to a nucleic acid sequence which can be assigned and introduced to an individual nucleic acid e.g., a cDNA molecule, and used to discriminate between individual nucleic acid molecules.
Similarly, terms such as “unique cell barcode sequence” and “unique tissue barcodes” will be understood to refer to unique sequences which, when introduced to a nucleic acid sequence e.g., such as during cDNA library construction, can be used to identify the cell or tissue (as appropriate) from which the nucleic acid sequences derives.
The term. “nucleic acid”, “nucleic acid molecule” and “polynucleotide” are used interchangeably herein to refer to a polymer having multiple nucleotide monomers. A nucleic acid can be single- or double-stranded; and can be DNA (e.g., cDNA or genomic DNA), RNA (e.g., mRNA, tRNA, rRNA, snRNA and/or ncRNA), or hybrid polymers (e.g., DNA/RNA). The term “nucleic acid” does not refer to any particular length of polymer. Rather, a nucleic acid may be any length e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, greater than 10,000 bases, greater than 100,000 bases, greater than about 1,000,000 or more bases composed of nucleotides.
The term “sequencing,” as used herein, refers to a method by which the identity of a consecutive stretch of nucleotides within a nucleic acid molecule is identified. That is, the identify of individual nucleotides within the nucleic acid molecule are identified which collectively provide the sequence of the nucleic acid or a part thereof. A number of methods and platforms are known in the art for sequencing of nucleic acid molecules which are described herein. For the purpose of the present disclosure, these may be conveniently divided into short-read sequencing methods and long-read sequencing methods based on the capabilities of the various methods and sequencing chemistries.
As used herein, a “short-read sequencing method” shall be understood to mean sequencing methods which are capable of producing single reads of up to 1000 bases, such as from about 35 bases to about 1000 bases. However, typically short-read sequencing method produce reads of 500 bases or less. Exemplary short-read sequencing methods are described herein and include next generation sequencing methods such as sequencing-by-hybridization, sequencing-by-synthesis, sequencing-by-ligation platform, combinatorial probe anchor synthesis, and ion semiconductor sequencing. However, chain termination (Sanger sequencing) may also be used to produce short read sequences. In one particular example, a sequencing-by-synthesis method using the Illumina platform is used to produce short read sequences. It also follows that “short-read sequence data” is data comprising and/or relating to sequences of about 35 bases to about 1000 bases in length, and typically 500 bases or less.
Conversely, a long-read sequencing method shall be understood to mean a method capable of producing sequence reads in excess of 1000 bases. Exemplary long-read sequencing methods are described herein and include nanopore sequencing and single molecule real time (SMRT) sequencing. Of course, it will be appreciated that read length achieved using a long-read sequencing method is also dependent on preparation of the nucleic acid molecule library e.g., cDNA library, not just the sequencing platform. For example, if a library is produced with an average fragment length of about 500 bases, then the average length of reads obtained from such a library will not exceed that length. However, library preparation aside, it will be appreciated that long-read sequencing methods are capable of producing long-read sequences e.g., over 1 Mb. It also follows that “long-read sequence data” is data comprising and/or relating to sequences in excess of 1000 bases in length, such as for example, between 1000 bases and 500 kb. Preferably, “long-read sequences” in the context of the invention are full length cDNAs.
As used herein, the term “demultiplex”, demultiplexing” or similar shall be understood to mean a step or process of separating or dividing individual sequence reads within multiplexed sequence data comprising multiple sequences into separate sequence files based on an index sequence tag introduced to each sequence during construction of sequencing libraries.
The term “contigs” as used herein, refers to contiguous regions of DNA or RNA sequence. “Contigs” can be determined by any number methods known in the art, such as, by comparing sequencing reads for overlapping sequences, and/or by comparing sequencing reads against a databases of known sequences in order to identify which sequencing reads have a high probability of being contiguous.
As used herein, “single cell capture” will be understood to mean a process of isolating single cells from a population of cells.
As used herein, the term “targeted enrichment” shall be understood to refer to a process by which the relative representation of a particular species, category or type of nucleic acid molecule i.e., a target, within a population of different nucleic acid molecules is increased. As described herein, target enrichment may be achieved using a range of methods known in the art, such as hybridisation capture e.g., using biotinylated probes configured to hybridise to the target nucleic acid which can then be retrieved using a biotin ligand, and/or by a depletion method which removes unwanted nucleic acid species. Targeted enrichment may also be performed in silico following sequencing, either as an alternative or in combination with enrichment prior to or during sequencing.
As used herein, the term “assembling”, “assembly” or similar shall be understood to refer to a process comprising alignment of multiple sequences based on consensus regions in order to form a longer sequence. For example, assembly of multiple sequences may be for the purpose of reconstructing a longer sequence of which the multiple sequences are component parts.
As used herein, the term “clonotype” in the context of T and/or B cell sequences shall be understood to mean a specific antigen receptor sequence derived from V(D)J recombination during somatic genome rearrangement of T and B cells, which can be used to infer shared lymphocyte clonality, or evolutionary relatedness of lymphocytes. The specific sequence may contain mutations, such as introduced vis somatic hypermutation of B cells following their activation by antigen recognition, and therefore the most similar germline V(D)J sequence may be used to define the clonotype of a mutated V(D)J sequence. Following V(D)J recombination, it is extremely unlikely that two cells descended from different lymphocytes will carry the same antigen receptor sequence or ‘clonotype’.
A method for high-throughput and multiplexed phenotyping and characterisation of single cells, said method comprising:
In one example, unique tissue barcodes are assigned and introduced to the nucleic acid molecules at step (a) to enable subsequent pooling and multiplex analysis of nucleic acid molecules derived from more than one tissue type and/or sample e.g., including samples from different subjects.
One exemplary method of the disclosure comprising steps (a) to (i) above is conveniently illustrated in
Prior to steps (a) to (i) above, a single cell capture step may also be performed. However, the method may also be conveniently performed on cells previously isolated. Exemplary methods for single cell capture which may be employed in the method of the disclosure include a droplet-based microfluidics platform, a flow cytometry platform, a plate-based platform, microwell-based platform or any combination thereof. However, any single cell capture method known in the art may be used and is contemplated herein. In one particular example, a droplet-based microfluidics platform is used for single cell capture, as illustrated in
As described herein, the library of nucleic acid molecules may contain any nucleic acid molecule type, such as selected from the group consisting of cDNA, genomic DNA, barcodes, cellular RNA (e.g., mRNA, tRNA, rRNA, snRNA and/or ncRNA) and combinations thereof. However, in one example the method is performed with cDNA molecules, as illustrated in
As described herein and illustrated in
There are a number of sequencing technologies available which may be employed for the short-read sequencing, such as, for example, the sequencing-by-hybridization platform from Affymetrix Inc. (Sunnyvale, Calif.), the sequencing-by-synthesis platforms from 454 Life Sciences (Bradford, Conn.), Illumina/Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), the sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif.), ion semiconductor sequencing (also referred to as Ion Torrent sequencing) from ThermoFisher Scientific, and combinatorial probe anchor synthesis (cPAS) using the MGI Tech Platforms from BGI (China). However, other platforms are available and may be used. Furthermore, combinations of these platforms may be employed to generate the first set of sequencing data. In one particular example, sequencing is performed using a sequencing-by-synthesis method e.g., such as exemplified in
The second library component is subjected to long-read sequencing e.g., this may be performed in parallel to the sequencing of the first library component described above. Any platform known in the art capable of sequencing reads in excess of 1000 nucleotides in length is contemplated and may be employed. However, exemplary long-read sequencing methods for use in the method described herein include nanopore sequencing developed for example, by Oxford Nanopore Technologies and single molecule real time (SMRT) sequencing (SMRT™ technology of Pacific Biosciences). In one example, the long-read sequencing method employed is a nanopore sequencing method. In another example, the long-read sequencing method is a SMRT sequencing method. In yet another example, a combination of nanopore sequencing and SMRT sequencing is used.
As illustrated in
Alternatively or in addition to the use of hybridisation capture of sequences or features of interest, targeted enrichment may comprise depleting unwanted sequences or features from the long-read component.
In accordance with any of the examples relating to targeted enrichment, the long-read component may be enriched for T and/or B cell receptor sequences.
It is also contemplated that target enrichment may be performed on the resulting sequence data i.e., by in silico mean. That is, one or more in silico steps may be performed to enrich the second set of sequence data for sequences of interest e.g., by actively selecting for sequences of interest or depleting the data set of unwanted sequences. It is contemplated that an in silico target enrichment may be performed in addition to target enrichment of the second library component. However, it is possible that only in silico enrichment is performed.
In addition to the target enrichment performed on the second library component and/or the second set of sequence data, equivalent target enrichment steps may also be performed on the first library component and/or first set of sequence data. Thus, in some example, both the first and second sets of sequence data are enriched for target sequences.
As illustrated in
As described herein and illustrated at (9) of
The demultiplexing of the second set of sequence data may be performed in a supervised fashion e.g., by matching or comparing the long-read sequences directly, allowing errors or not. Alternatively, an unsupervised method may be employed to demultiplex the second set of sequence data, such as by comparing the long-read sequence data to itself in a manner sufficient to identify commonalities in long-read sequences or data that is associated with discriminating sequence features e.g., UMIs, cell barcodes, tissue barcodes etc.
Once the second set of sequence data is demultiplexed, molecular profile information relevant to the individual long-read sequences can be inferred based on molecular profiles characterised for corresponding sequences in the first set of sequence data at (d), wherein corresponding sequences from the first and second sets of sequence data are identified using the UMIs, unique cell barcodes, unique tissue barcodes, or a combinations thereof. The individual long-read sequences can then be organised into one or more groups based on information commonly assigned to each sequence e.g., information relating to cell type, tissue type, genes, sequences, molecules and other discriminative features described herein. Organisation of the demultiplexed long-read sequences into one or more groups may comprise de novo assembly of long-read sequences, alignment of long-read sequences to one or more reference sequences, multiple sequence alignments or other approach capable of grouping the long-read sequences e.g., based on tissue type, cell type and/or sequence type. Within each group, long-read sequences can then be assembled and one or more contigs produced based on consensus sequence. Any suitable software known in the art for long-read assembly may be employed. In particular one example, de novo grouping and assembly of the demultiplexed long-read sequences into one or more contigs is performed using CANU software (Koren et al., (2017) Genome Research, 27:1-15. In accordance with an example in which 10× Genomics platform is used to prepare barcoded cDNA libraries, the GemCode may also be used to perform de novo assembly of grouped long-read sequences into contigs. This step is illustrated at (10) of
As illustrated at step (11) of
Molecular characterisation of the contigs may then be undertaken. In one example, the molecular characterisation of the contigs comprises characterisation of contigs on the basis of one or more of the following: antigen receptor clonotyping, mutation analysis, somatic genome variation, alternative transcript splicing, fusion genes or chimeric transcripts, transcript isoform quantification and combinations thereof. In one example, molecular characterisation of the contigs may be performed using IgBlast. IgBlast may be particularly useful in circumstances in which the second set of sequence data has been enriched for T and/or B cell receptor sequences, either prior to long-read sequencing (as illustrated in
Molecular characterisation of the contigs may also be based on one or more of the following:
The disclosure also provides a computer implemented method for phenotyping and characterising single cells using data obtained from high-throughput and multiplexed long-read single cell sequencing, comprising:
In one example, each nucleic acid molecule comprises a unique tissue barcode to enable pooling and deconvolution of sequence data for nucleic acid molecules derived from more than one tissue type and/or sample e.g., including samples from different subjects.
As described herein, the library of nucleic acid molecules may contain any nucleic acid molecule selected from the group consisting of cDNA, genomic DNA, barcodes, cellular RNA (e.g., such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA) and/or non-coding RNA (ncRNA)) and combinations thereof. In one example, the library of nucleic acid molecules comprises cDNA. In one example, the library of nucleic acid molecules comprises genomic DNA. In one example, the library of nucleic acid molecules comprises barcodes. In one example, the library of nucleic acid molecules comprises cellular RNA e.g., such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA) and/or non-coding RNA (ncRNA). In one example, the library of nucleic acid molecules comprises a mixture of cDNA, genomic DNA, barcodes and cellular RNA.
The first set of sequence data may be generated by a short-read sequencing method and/or a long-read sequencing method. In one example, the first set of sequence data has been generated using a short-read sequencing method. In one example, the first set of sequence data has been using a long-read sequencing method. In yet another example, the first set of sequence data has been using both short and long-read sequencing methods. Short-read sequencing methods have been described herein and shall be taken to apply mutatis mutandis to each and every example describing computer implemented methods In one particular example, the first set of sequence data was generated by a method employing a sequencing-by-synthesis method e.g., as illustrated in
Computational analyses may be performed on the first set of sequence data to reveal discriminative features of the sequence population through single cell molecular profiling e.g., discriminative features may include, but are not limited to genetic, epigenetic and/or transcriptomic features capable of distinguishing between single cells.
As described herein, the second set of sequence data received may be generated by any platform known in the art capable of consistently sequencing and transmitting reads in excess of 1000 nucleotides in length. Exemplary long-read sequencing technologies have already been described herein and shall be taken to apply mutatis mutandis to each and every example describing computer implemented methods. In one particular example, the second set of sequence data received has been generated using a nanopore sequencing method.
The second set of sequence data may be enriched for genetic, epigenetic or transcriptomic sequences or features or interest i.e., to ensure that sequences of interest are appropriately represented in the data. The first set of sequence data may also be enriched for the genetic, epigenetic or transcriptomic sequences or features or interest i.e., to ensure that sequences of interest are appropriately represented in the data. In one example, the first and/or second sequence data sets may have been produced using a method whereby target enrichment was performed prior to sequencing. Alternatively, or in addition, the first and/or second sequence data sets may have been produced using a method whereby target enrichment was performed post sequencing. In the case of the latter, the computer implemented method may comprise performing one of more in silico steps to enrich the first and/or second sets of sequence data for sequences or features of interest. Examples of target enrichment have been described herein and shall be taken to apply mutatis mutandis to each and every example describing computer implemented methods. In one particular example, the second set of sequence data is enriched for T and/or B cell receptor sequences.
The computer implemented method may comprises performing one or more filtering step on the second set of sequence data to remove sequences which are, for example, of undesired length (e.g., <500 bases long), uninformative, erroneous and/or not of interest. This may also assist in enriching the second set of sequence data for sequences of interest. Appropriate programs for computational filtering of sequence data will be known to a person skilled in the art. Filtering may also involve removing adapter sequences which were added during preparation of nucleic acid molecules libraries e.g., cDNA libraries. The filtering step may be performed on the second set of sequence data at any time prior to de novo assembly into contigs. For example, as illustrated in
The computer implemented method described herein involves a demultiplexing step in which the second set of sequence data is separated into its component sequences. Further, unique cell barcode and UMI sequences, and optionally unique tissue barcodes (if applicable), assigned and introduced to the nucleic acid molecules e.g., cDNA molecules, during library construction, are identified in the long-read sequences to characterise the respective long-read sequences. Demultiplexing also involves comparing or matching the long-read sequences to the corresponding sequences in the first set of sequence data using the UMI and unique cell barcodes and optionally unique tissue barcodes. Thus, discriminative features of the sequences in the first set of sequence data identified through molecular profiling at step (d) may be inferred for the corresponding, matched long-read sequences. These discriminative features may include, but are not limited to genetic, epigenetic and/or transcriptomic features capable of distinguishing between single cells. Matching of long-read sequences to corresponding sequences in the first set of sequence data can also be used as a further filter to identify and correct potential errors in the long-read sequences.
The demultiplexing of the long-read sequence data may be performed in a supervised fashion e.g., by matching or comparing the long-read sequences directly, allowing errors or not. Alternatively, an unsupervised method may be employed to demultiplex the long-read sequences, such as by comparing the second set of sequence data to itself in a manner sufficient to identify commonalities in long-read sequences or data that is associated with discriminating sequence features e.g., UMIs, cell barcodes, tissue barcodes etc.
Once the second set of sequence data is demultiplexed, molecular profile information relevant to the individual long-read sequences can be inferred based on molecular profiles characterised for corresponding sequences in the first set of sequence data at (b), wherein corresponding sequences from the first and second sets of sequence data are identified using the UMIs, unique cell barcodes, unique tissue barcodes, or a combinations thereof. The individual long-read sequences can then be organised into one or more groups based on information commonly assigned to each sequence at the demultiplexing step e.g., information relating to cell type, tissue type, unique molecules, sequence of interest and other discriminative features described herein. Organisation of the demultiplexed long-read sequences into one or more groups may comprise de novo assembly of long-read sequences, alignment of long-read sequences to one or more reference sequences, multiple sequence alignments or other approach capable of grouping the long-read sequences e.g., based on tissue type, cell type and/or sequence type. Within each group, long-read sequences can then be assembled and one or more contigs produced based on consensus sequence. Any suitable software known in the art for long-read assembly may be employed. In particular one example, de novo grouping and assembly of the demultiplexed long-read sequences into one or more contigs is performed using CANU software (Koren et al., (2017) Genome Research, 27:1-15. In accordance with an example in which 10× Genomics platform is used to prepare barcoded cDNA libraries, the GemCode may also be used to perform de novo assembly of grouped long-read sequences into contigs. This step is illustrated at (10) of
The computer implemented method may also optionally comprise one or more correction and/or polishing steps to correct errors in the long-read sequences and/or contigs and thereby improve the consensus sequence. As described herein, the one or more correction and/or polishing steps may be performed at multiple stages of the method e.g., prior to demultiplexing, post-demultiplexing, post contig assembly. For example, contig consensus correction may be performed using Minimap2 and/or RACON. For example, consensus polishing may be performed using Minimap2 and/or Nanopolish. However, any software programs known to be useful for correction and/or polishing of sequences may be employed and are contemplated for use herein.
Molecular characterisation of the contigs may then be undertaken. In one example, the molecular characterisation of the contigs comprises characterisation of contigs on the basis of one or more of the following: antigen receptor clonotyping, mutation analysis, somatic genome variation, alternative transcript splicing, fusion genes or chimeric transcripts, transcript isoform quantification and combinations thereof. In one example, molecular characterisation of the contigs may be performed using IgBlast. IgBlast may be particularly useful in circumstances where an enrichment has been performed for T and/or B cell receptor sequences (as illustrated in
Molecular characterisation of the contigs may also be based on one or more of the following:
This example describes a rapid high-throughput method for sequencing full length transcripts using targeted capture and Oxford nanopore sequencing, and linking this with short-read transcriptome protein epitope profiling at single cell resolution. This novel method, termed Repertoire and Gene Expression by sequencing (RAGE-seq), may be applied to high-throughput droplet-based scRNA-seq workflows to accurately pair gene expression profiles with targeted full length cDNA sequences from a large number of cells.
In addition to describing the method, this example demonstrates the power of RAGE-seq by generating full transcriptome and full length sequence for antigen receptors and PTPRC (encoding CD45) from thousands of human tumour-associated lymphocytes. Using de novo assembly of nanopore reads, clonotype sequences were recovered at high accuracy and sensitivity, including the accurate calling of somatic mutations from full-length IgH and IgL chains allowing for the inference of B cell clonal evolution. Furthermore, PTPRC splice variants encoding alternate isoforms of CD45 were determined by targeted capture and long-read sequencing, providing important information on whether lymphocytes were naive (CD45RA) or antigen-experienced (CD45RO).
Finally, it is shown that RAGE-Seq is uniquely compatible with Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) (doi:10.1038/nMeth.4380), a remarkable new method that permits simultaneous measurement of transcriptomes and protein epitopes at single cell resolution. The combination of RAGE-Seq with CITE-Seq affords extremely high resolution multi-omic analysis of cellular phenotype and transcriptional output.
Droplet-microfluidics form one of the most commonly used high-throughput single-cell RNA-sequencing methods due to their fast encapsulation of a large number of individual cells and nanolitre reaction volume. Typically, full-length cDNA libraries generated from these platforms undergo fragmentation and PCR enrichment of the 3′ end of molecules containing the cell barcode so they are suitable for short-read sequencing. In contrast to existing methods, the inventors designed a strategy which involved splitting full-length single-cell 3′-tag or 5′-tag cDNA libraries prior to fragmentation for short-read sequencing, and selectively enriching BCR and TCR transcripts using targeted hybridization capture. Targeted capture was chosen over commonly used PCR methods (Carlson et al., (2013) Nature communications, 4:2680; Shugay et al., (2014) Nature methods, 11(6):653-5) to retain full-length transcripts. Enriched antigen-receptor molecules were then subjected to long-read Nanopore sequencing to obtain both the 3′ or 5′ cell-barcode and the 5′ VDJ sequence. In parallel, short-read Illumina sequencing was performed to profile gene expression on the remaining cDNA. By matching the cell barcodes obtained from long-read sequencing with the cell barcodes obtained from Illumina sequencing, transcriptome profiles of individual cells could be linked with clonotype sequence (
The inventors also designed a capture bait library with biotinylated probes specifically targeting all annotated and functional human V, J and Constant (C) region exons within the genomic loci that encode TCRα, TCRβ, TCRd, TCRg, IgH, Igκ and Igλ chains. Capture probes were chosen over V-region specific PCR primers to minimize preferential bias introduced during multiplex PCR (Carlson et al., 2013) and to retain full-length transcripts. The capture bait library also included probes specific for the PTPRC transcript, encoding the CD45 protein. Activation of lymphocytes by antigen causes a switch in PTPRC splicing, resulting in the expression of unique CD45 proteins on the cell surface. These can be used to distinguish naive from antigen-experienced lymphocytes and so are very informative in the study of lymphocyte biology. However, PTPRC splice variants cannot be accurately measured using short-read sequencing.
Oxford Nanopore Technologies (ONT) sequencing was chosen to read full-length cDNA molecules due to its high-throughput and low cost. The major challenge of generating highly accurate clonotype sequences with long-read technologies, however, is the high error rate, estimated at ˜10% error per nucleotide. Whole genome assemblies generated from long-read sequencing often overcome this error limitation through the use of de novo assembly followed by ‘polishing’ to achieve high accuracy of over 99% (Jain M., et al. (2018) Nature Biotech). It was predicted that such approaches could also be applied to Nanopore reads generated from cDNA targeted capture and a computational pipeline was developed that performs de novo assembly on demultiplexed Nanopore data to generate full-length clonotype sequences for each cell (
The RAGE-seq pipeline is more fully illustrated in the flowchart presented in
Patient tissues used in this work were collected under protocol X13-0133, HREC/13/RPAH/187. HREC approval was obtained through the SLHD (Sydney Local Health District) Ethics Committee (Royal Prince Alfred Hospital zone), and site-specific approvals were obtained for all additional sites. Written consent was obtained from all patients prior to collection of tissue and clinical data stored in a de-identified manner, following pre-approved protocols. Tissue analysis was performed under protocol x14-021, LNR/14/RPAH/155.
Following surgical resection of tumour and lymph node from patient, samples were transferred in ice cold RPMI-1640 with 50% FCS to the laboratory to be processed. Tumour was cut into approximately 1 mm3 pieces and dissociated as per MACS human tumour dissociation kit (Miltenyi Biotec, Australia). Lymph node was similarly processed however with digestion halted at 15 minutes. After washing twice with 2% FBS in PBS, cells were resuspended in sorting buffer and passed through 70 um strainers. The Jurkat T-cell line and Ramos B-cell line were cultured in RPMI-1640 medium with 10% FCS. Monocytes were flow sorted from human peripheral blood mononuclear cells (PBMCs) using a human anti-CD14 antibody. Flow cytometric sorter (BD FACS AriaIII) was used enrich for viable cells using DAPI stain, maintaining a gating threshold which omits red blood cells. Cells were centrifuged and resuspended in PBS with 2% FCS to obtain an approximate concentration of 1000 cells/μ1 which was counted using a haemocytometer. Samples were always handled on ice when possible and a viability of at least ˜90% for all samples was confirmed by trypan blue stain prior to capture.
1.2.3 Droplet Based scRNAseq (10× Genomics) Capture was performed as per 10× Chromium Single Cell 3′ (V2 chemistry) protocol, aiming for an estimate of 4000 captured cells for each sample. Full-length cDNA was split 1:1 for Nanopore long-read sequencing and for short read sequencing. An Illumina NextSeq 500 was used to sequence the transcriptome library, and the yielded raw bcl file was demultiplexed and aligned (hg38 build) using CellRanger 2.0 (10× Genomics).
A target enrichment library (Roche NimbleGen) was designed by first identifying gene annotations of all functional V (IGHV, IGKV, IGLV, TRAY, TRBV, TRGV), J (IGHJ, IGKJ, IGLJ, TRAJ, TRBJ, TRGJ, TRDJ), and C (IGHA, IGHD, IGHE, IGHG, IGHM, IGKC, IGLC, TRAC, TRBC, TRGC, TRDC) and PTPRC genes obtained from the IMGT database [doi: 10.1093/nar/gku1056]. For each gene, genome coordinates of their corresponding exons were obtained from the GRCh38 primary assembly. Design of probes from target regions and synthesis was performed by Roche NimbleGen using the SeqCap RNA Choice format with a maximum of 5 matches to the human genome. 66 regions were removed from the final design due to being too small according to the NimbleDesign tool. In total 678 exons were targeted by the CaptureSeq array targeting ˜128 Kb.
Following full-length cDNA amplification one tenth to one half of the total volume of cDNA was used for targeted capture and nanopore sequencing. Pre-capture PCR was first performed using KAPA-Hi-Fi polymerase, 3 mM TSO and 3 mM R1 primer with the following cycling conditions: 98° C. for 3 min; 98° C. for 20 s, 65° C. for 30 s, 72° C. for 1 min 30 s×5 cycles (cell lines) or ×20 cycles (primary cells). Next PCR products were purified using AMPure XP beads and 500 ng-1 ug of amplified cDNA was used for targeted capture using the protocol previously described (Mercer et al., (2014) Nature protocols, 9(5):989-1009), with the following modifications. Universal hybridisation enhancing (HE) oligo and index HE oligos were not included during hybridisation. Two rounds of hybridisation were performed for 24 h each at 47° C. Following each round of hybridisation and capture, PCR was performed using KAPA hi fi instead of Phusion DNA polymerase with 1 mM TSO primer and 1 mM R1 primer (instead of the TS-PCR oligos) with the following PCR cycling conditions: 98° C. for 3 min; 98° C. for 20 s, 65° C. for 15 s, 72° C. for 1 min 30 s×5 cycles (first round) or ×20 cycles (second round). Postcapture cDNA library size ranged from 0.6 to 2 kb.
SmartSeq2 was performed as described by Picelli et al., (2014) Nature protocols, 9(1):171-81 with the following modifications: the IS PCR primer was reduced to a 50 nM final concentration and the number of PCR cycles increased to 28. Sequencing was performed on the Illumina NextSeq platform.
1.2.7 scRNAseq Count Matrix Processing
The raw gene expression matrices were normalised and scaled using Seurat (v3.4) (Satija et al., (2015) Nature biotechnology, 33(5):495). For the cell line capture, cells that express <250 genes or <1000 UMIs or that contain more than 6% UMIs derived from mitochondrial genome were excluded. To reduce doublet contamination, any cells expressing >6500 genes were discarded, additionally removing any cells that are ×5 deviated from the median gene count for that cell type. For lymph node and tumour, a threshold of <100 genes or <500 UMIs was set to allow detection of exhausted T-cells. A principle component analysis was performed on the variable genes and by using the Jackstraw method, the first principle components with a P-value<0.01 was used for dimensional reduction. For Tumour and Lymphnode combined analysis, Seurat's RunCCA was used for cross-dataset normalization to enable subsequent comparative analyses. Cell cycle scoring was performed using scRNA cell cycle gene expression scores from Nestorowa et al., (2016) Blood, 128(8): e20-31 and Tirosh et al., (2016) Science, 352(6282):189-96.
The resolution set for each tSNE analysis was determinant on the strength of annotation using well known canonical marker genes and Seurat's FindAllMarkers function yielding an average expression for any particular cluster which yielded >2.5-fold higher than the average expression in other sub clusters from that cell type.
Hybridisation capture cDNA libraries were prepared for long read sequencing using Oxford Nanopore Technologies' (ONT) 1D adapter ligation sequencing kit (SQK-LSK108), with the exception of one sample that used the 1D2 adapter ligation kit (LSK-308). The latter was base called and considered as 1D for all subsequent steps. All samples were sequenced with R9.4.1 flowcells (FLO-MIN106), with the exception of 3/6 cell line samples that were loaded onto R9.5.1 (FLO-MIN107) flowcells (including the aforementioned LSK308 sample). Base calling was performed offline on a high-performance computing cluster using ONT's Albacore software pipeline (version 2.2.7). A list of samples, chemistries, flowcell identification numbers, and manufacturer software versions can be found in Table 1.
Base called fastq files were pooled for each biological sample and subjected to ad hoc demultiplexing using a direct sequence matching strategy (i.e. 0 mismatches and indels). Cell barcode sequences (16 nt) were extracted from matched short read sequencing data, as produced by 10× Genomic's CellRanger software. Forward and reverse-complemented cell barcode sequences were then used to demultiplex the nanopore sequencing reads. This was achieved by scanning the first and last 200 nt of any read longer than 250 nt for an exact match to the list of barcodes (10× genomics). The reads were trimmed by ‘chopping’ the read 13 nt downstream of the position matching a cell barcode to ensure that (i) the 10 nt UMI sequence is removed from consensus assembly steps, and (ii) potential insertions are also removed, which may also remove a few bases of the poly-T/Ail. The fastq headers were modified to include barcode and UMI sequences post-demultiplexing.
As highlighted in
Polished fasta files containing consensus transcript contigs for each cell barcode were subjected to IgBLAST (Ye et al., (2013) Nucleic acids research, 41(Web Server issue):W34-40) alignment to determine V(D)J rearrangements and blastn alignment (Camacho et al., (2009) BMC bioinformatics, 10:421) to determine the Ig or TCR constant regions exons associated with the V(D)J. For each contig, separate IgBLAST for immunoglobulin and TCR were performed using IMGT germline gene reference datasets (Lefranc et al., (2015) Nucleic acids research, 43(Database issue):D413-22). Amino acid sequences and location of CDR3 were defined by the conserved cysteine-104 and typtophan-118 based on the IMGT numbering system (Lefranc et al., (2015) Nucleic acids research, 43(Database issue):D413-22). IgBLAST parameters were default with the exception of returning only a single gene segment per V(D)J loci. Text-based IgBLAST output was then parsed to tab-delimited summaries, calling gene segments, framework and complementarity determining regions, mismatches, and indels relative to germline gene segments. Following this first round of IgBLAST, insertions and deletions (indels) in parts of the sequence that aligned to germline gene segments were corrected to their closest germline gene, and the IgBLAST step was repeated to generate indel corrected alignments. This was particularly important for correction of Nanopore sequencing errors that would otherwise impact on the reading frame of the V(D)J rearrangement that would prevent the CDR3 from being determined accurately. The impact of correcting insertions/deletions (indels) is shown in
Clonotypes that were out-of-frame or that contained stop codons, termed non-productive clonotypes, were removed unless stated otherwise. BCR clonotypes containing more than 40 mutations or TCRs with more than 5 mutations in their respective V gene segments were filtered against. Analysis of SHM of Jurkat V regions included no filtering of clonotypes based on number of mutations (
Several methods for isoform assignment can be employed from the alignments of either raw reads or consensus transcript contigs to the canonical CD45 splice variants. For this example, depth of coverage and coordinates of the raw reads for each cell barcode that span exon 1-7 can be used to assign likelihood of belonging to a particular CD45 isoform. CITE-Seq data can be used to validate isoform assignment.
Of the cells that aligned to canonical CD45 splice variants, cells that were missing either exon 3 or exon 7 of CD45 were removed (all known CD45 isoforms should include these exons). Cells with less than 50 reads were removed.
To determine the spliced constant regions exons that were associated with the V(D)J rearrangement blastn was used to align each contig against the spliced reference exons. For the IGHC, both the membrane and secreted versions of each constant region were included. Tabular blastn output was parsed to call constant region for each contig using the criteria of greater than 95% coverage of the spliced constant region exons and percentage identity of more than 90%. A 90% identity threshold was used as contigs used for constant region calling were not indel corrected.
1.2.17 Integration of Clonotype with scRNA-Seq
Clonotypes that define groups of cells that are likely to have arisen from clonal expansion of the same progenitor B or T cell were defined either be shared gene rearrangements using the same V and J germline gene segments with identical CDR3 amino acid sequences for the T cells, and same V and J germline gene segments with 90% identical CDR3 nucleotide sequence for B cells. Clonotypes either shared the same paired chains (e.g., heavy and light chains for BCR, and alpha/beta or gamma/delta chains for the TCR) or shared TCRβ or IGH chains.
Read subsampling was performed on 200 Jurkat and 200 Ramos cells with each cell having no less than one thousand reads. The subsampling itself was performed with the sequence analysis toolkit, seqtk version 1.0-r72 (https://github.com/lh3/seqtk), using the sample command with a seed parameter of −s123. Subsampling was performed in a stepwise manner at increments of 1000, 500, 250, 100 and 50 read depths, with the resulting subsampled fastq the next input in later rounds of subsampling.
Alignment of nanopore reads to TCR and BCR genes was performed by the alignment program Minimap2 version 2.3-r536 (Li H. (2009) Bioinformatics (Oxford, England)) to a custom reference fasta sequence containing TCR and BCR constant region genes, using the ‘-x map-ont’ preset. The resulting alignments were sorted and then viewed using samtools (Li et al., (2009) Bioinformatics (Oxford, England), 25(16):2078-9) version 1.7-2-gc6125d0 (with htslib 1.7-6-g6d2bfb7) and reads flagged as unmapped, not primary or supplementary were not counted as on-target.
Nanopore reads were aligned to the CD45 primary assembly (GRCh38.p12) as previously described, then aligned to a custom reference fasta sequence containing CD45 exon sequences that discriminate into canonical CD45 splice variants (i.e. RABC, RO, etc.). Depth of coverage across these splice variant sites was examined using Samtools version 1.7-2-gc6125d0 (with htslib 1.7-6-g6d2bfb7) via the depth command.
CITE-Seq is a method to permit simultaneous measurement of transcriptomes and cell surface epitopes using barcoded antibodies. (doi:10.1038/nMeth.4380). Essentially as described, cell suspensions were stained with a pool of 87 uniquely barcoded antibodies (purchased from Biolegend Inc) prior to capture using the 10× genomics system. Following size fractionation of cDNAs, CITE-Seq libraries were sequenced separately using an Illumina Next-Seq platform and analysed using Seurat.
To assess the validity of this method, the inventors then performed RAGE-Seq on a mixture of the human T cell line Jurkat and the human B cell line Ramos, for which antigen receptor sequences are published (
To demultiplex 10× cellular barcodes from the nanopore sequencing reads, a whitelist of cell barcodes from Illumina sequencing was generated and used to search for a direct match within each read. Using this approach, 3,805,076 de-multiplexed reads (18.7%) containing all of the 10× cell barcodes (
The inventors also carried out de novo assembly, error correction and contig polishing on the nanopore reads (see Methods), generating on average 4.26 contigs per Jurkat cell, 5.24 per Ramos cell and 0.12 per Monocyte (
For Jurkat cells, paired TCRα and TCRβ chains were recovered from 18.9% of cells, 13.3% with a TCRα chain only and 39.6% with a TCRβ chain only (
Next, the accuracy of calling a correct clonotype at nucleotide resolution was evaluated by investigating the CDR3 region of Jurkat cells against their known reference CDR3 sequences (
RAGE-Seq was also compared against the reconstruction of Jurkat TCR sequences produced using SmartSeq2 using VDJ-Puzzle (Eltahla et al., (2016) Immunology and cell biology, 94(6):604-11). VDJ-Puzzle was able to recover more TCR clonotypes at high accuracy. However, RAGE-Seq proved to be ˜30 times more cost effective on a per cell basis (Table 2). Taken together, these results indicate that RAGE-Seq is both accurate and sensitive in determining clonotype sequences and has significant advantages over SmartSeq2 in terms of cost and throughput.
B cells can acquire additional BCR diversity through somatic hypermutation (SHM) of variable regions of immunoglobulin genes. The Ramos cell line is known to mutate its receptors by undergoing SHM in culture (Sale and Neuberger (1998) Immunity, 9(6):859-69). To assess the impact of SHM on BCR diversity, accurate sequence across the entire V region of the heavy and light chain is required. Here, RAGE-Seq was able to recover over 99% of Ramos IgH and IgL clonotypes with the complete V region length (
Jurkat TRAV (TCRα) and TRBV (TCRβ) genes were then interrogated to assess the accuracy of RAGE-seq to call SHM, which should be completely conserved in this clonal cell line. A low number of Jurkat cells with one or more nucleotide mismatches to germline in these regions were identified (TRAV: 5.05%, TRBV: 2.8%,
RAGE-Seq was then performed on a human lymph node resected from a breast cancer patient in order apply the method to primary B and T lymphocytes. In doing so, 4,165 T cells were identified which could be subdivided into 6 clusters: CD4 effector memory (EM; 1069 cells), CD4 central memory (CM; 1321 cells), CD4 T follicular cells (TfH; 142 cells), CD4 T regulatory cells (Treg; 740 cells), CD8 CM (487) and CD8 effector (EF)/NKT (405 cells) (
The targeted capture panel included probes against TCRγ and TCR allowing for the detection of TCRγδ cells, a poorly-explored class of unconventional T cell, of substantial interest to studies of infection and tumour immunology. A total of 11 T cells in the lymph node were assigned paired TCRγδ chains, the majority of which clustered in the CD8 EFF cluster. 92 T-cells were recovered with only the TCRγ chain and 14 T cells with only the TCR chain only, again the majority of which clustered in the CD8 EFF population (
Upon activation, B cells can change their antibody effector function through genome rearrangements in the BCR heavy chain constant region, known as isotype class switching (Di Noia and Neuberger (2007) Annual review of biochemistry, 76:1-22) and also generate membrane-associated or secreted immunoglobulins via alternative splicing (Alt et al., (1980) Cell, 20(2):293-301). Naïve B cells predominantly express BCR transcripts that are non-mutated and express both IGHM and IGHD isotypes. Upon activation, however, B cells mutate their BCR and can replace IGHM with IGHG, IGHE or IGHA isotypes (Alt et al., (1980) Cell, 20(2):293-301); Chaudhuri and Alt (2004) Nature reviews Immunology, 4(7):541-52). Alternative splicing controls the expression of IGHD and IGHM, but also membrane-form and secreted-form transcripts of IgH, which transitions as B cells becoming antibody secreting cells. As expected, memory B cells in the lymph node were more mutated, had undergone isotype switching and had a greater number of IgH clonotypes assigned the secreted-form when compared to naïve B cells (
Clonal expansion in the lymph node was uncommon, with B or T cell clones only detected in a maximum of two cells within the total population. For B cells there were 13 expanded clones, the majority of which segregated in the naïve B cell cluster, while for T cells there were also 13 expanded clones which clustered by cell type (
An important application of RAGE-Seq is the ability to track clonally related T or B cells across tissues, to gain systems-level insights into the evolution of immune responses. One such application is the analysis of lymphocytes in a tumour and its draining lymph node, the presumptive site of antigen presentation and source of tumour-infiltrating lymphocytes (TILs). The inventors therefore performed RAGE-Seq on a patient-matched primary tumour and compared the results to the lymphocytes found in the patient's lymph node. From a total of 2493 captured cells, 909 T cells and 215 B cells (
To investigate whether clonally related cells have common gene expression features across tissues, more stringent thresholds for clonality were applied, analysing lymphocytes expressing paired receptor chains or the highly diverse TCRβ and IGH. Seven shared clones were identified, six of them within the CD8 EFF cluster (
The presence of clonally expanded T cells between tissues suggested that these cells were proliferating in response to antigen stimulation. To examine this further, the scRNA-Seq data was used to perform cell cycle analysis of all cells within each CD8 (EFF) cluster of tumour and lymph node to infer whether TIL persistence of the clone is through proliferation occurring at the site of each sample and/or through trafficking between tissues. This was additionally performed on any expanded clones in tumour not shared with lymphnode (
To determine whether RAGE-Seq is compatible with other multi-omic methods, the inventors analysed an additional metastatic lymph node sample (metastatic triple negative breast cancer) using RAGE-Seq together with CITE-Seq, a method to simultaneously determine transcriptome and protein epitope data in thousands of single cells. Cells were stained with a panel of 87 uniquely barcoded antibodies against immune and tumour markers and immune checkpoint molecules and partitioned using the 10× chromium system. A total of 3113 cells were captured using the 10× Chromium platform and subjected to RAGE-Seq, capturing TCR, BCR and PTPRC (the gene encoding CD45). RNA and CITE-Seq libraries were separated by size fractionation and sequenced separately by illumina short-read sequencing while targeted capture long read sequencing using the nanopore platform was conducted for PTPRC (encoding CD45) and all TCR and BCR genes, as described earlier. The data presented in
Pairing the clonotype of a BCR or TCR with a functional phenotype of a B or T cell offers great insights into B and T cell responses. RAGE-Seq has been shown to be robust in its ability to sample across both Illumina and Nanopore sequencing platforms and highly sensitive and accurate in providing full-length BCR and TCR sequences across immortalized and primary human B and T cells. Given its greater throughput and substantially lower cost, RAGE-Seq has significant advantages over SmartSeq2 for immune profiling. As a result, RAGE-Seq can circumvent the need to isolate specific lymphocyte populations by flow cytometry, permitting retrospective characterization of low abundance lymphocytes within tissues. As shown herein, using RAGE-seq it was possible to identify clones with unique gene expression features that had expanded and were shared across tissues, despite unbiased sampling from a breast cancer, which generally have low TIL frequency.
In this study the inventors demonstrate the compatibility of RAGE-Seq with the 10× Chromium 3′ and/or 5′ system. However, the RAGE-seq pipeline may be adapted to any high-throughput single cell RNA-sequencing technologies that employ 3′ and/or 5′ cell-barcode tagging. Furthermore, a number of these methods are compatible with current DNA barcoded antibody technologies CITE-Seq and REAP-Seq (Stoeckius et al., (2017) Nature methods, 14(9):865-8; Peterson et al., (2017) Nature biotechnology, 35(10):936-9; Shahi et al., (2017) Scientific reports, 7:44447), which are powerful tools for immunophenotyping, allowing the additional measurement of cell surface proteins. The combination of RAGE-Seq with CITE-Seq permits the simultaneous phenotyping of cellular populations using protein targets plus RNA with full length sequencing capacity. This will be hugely valuable to numerous areas of investigation, including tumour immunology, autoimmunity, functional genomics or clonal evolution in cancer. It can be applied more broadly to any study in which the incorporation of feature barcoding, such as CITE-Seq or CRISPR barcodes, adds value to RAGE-Seq. The inventors anticipate that the high nucleotide accuracy achieved by RAGE-Seq will be applicable to identifying somatic variants in individual cancer cells and link this with gene-expression profiles.
Recently, the commercially available Single Cell V(D)J+5′ Gene Expression kit has been used to profile TILs in Breast cancer (Azizi et al., (2018) Cell, 174(5):1293-1308), relying on the incorporation of cell barcodes on the 5′ end of mRNA transcripts and VDJ-specific PCR amplification. Compared to this method, RAGE-Seq has several advantages:
1) it is compatible with CITE-Seq and REAP-Seq and sequences receptors from all lymphocytes in a single reaction, including gd T cells which are of increasing interest in infection and cancer immunology (Zhao et al., (2018) Journal of translational medicine, 16(1):3);
2) RAGE-Seq provides full length receptor sequence, which is essential in the analysis of immunoglobulin SHM; and
3) the recovery of paired full length IgH and IgL sequences also allows for the synthesis of recombinant antibodies which can be used to explore the antigen specificity of B cells of interest.
A further advantage of RAGE-Seq is the ability to detect splice isoforms at the single cell level, which has been demonstrated herein by detecting IgH isoforms destined for antibody secretion or membrane-integration. To the inventors' knowledge, this is the first report integrating IgH V(D)J clonotype sequences with analysis of membrane or secreted exons.
Whilst the focus in this proof of concept study is on lymphocyte receptors, the inventors anticipate that any transcripts could be targeted using this method, simply by changing the composition of the capture probe library. This may include panels targeting cancer driver genes, genes controlling or regulated by splicing, pathogenic fusion genes or genes that are otherwise difficult to detect using short-read sequencing, In this regard, RAGE-seq is a generalisable experimental and computation pipeline to integrate gene expression with targeted analysis of splicing, structural variation and somatic mutation from thousands of single cells. One can envisage this method being applied to multiple areas of biology, such as oncology where RAGE-Seq could be used to track the transcriptional consequences of clonal evolution at single cell resolution. Similarly, RAGE-seq could be applied to neurobiology where alternative splicing and somatic retrotransposition into genes drive brain development and disease (Baillie et al., (2011) Nature, 479(7374):534-7). The adaptability of RAGE-Seq across multiple scRNA-seq platforms and the flexibility to target a range of genes of interest may be of particular value in comprehensively describing a human cell atlas.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018903546 | Sep 2018 | AU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2019/050101 | 2/8/2019 | WO | 00 |
