Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

• Certain aspects of the disclosure are directed to a method of co-amplifying genomic DNA (gDNA) and RNA from a single biological sample, the method comprising: a) lysing a biological sample to release a plurality of nucleic acids comprising both gDNA and RNA from the biological sample; b) fragmenting the gDNA in the plurality of nucleic acids; c) attaching a DNA adaptor to the fragmented gDNA from (b) to form a plurality of DNA fragmentadaptor molecules; d) synthesizing complementary DNA (cDNA) from the RNA (e.g., unfragmented RNA) in the plurality of nucleic acids, wherein the synthesizing comprises reverse transcription comprising a reverse transcriptase and an RNA primer, wherein the RNA primer comprises an RNA adaptor which is distinguishable from the DNA adaptor to form a plurality of cDNA-adaptor molecules; and e) co-amplifying the plurality of DNA fragment-adapter molecules and the plurality
• the single cell or plurality of cells comprise a live cell, a genetically engineered cell, a perturbed cell, or a fixed cell.
• the biological sample comprises a micro-dissected tissue (e.g., fresh or fixed). In some aspects, the micro-dissected tissue is fresh. In some aspects, the micro-dissected tissue is fixed. In some aspects, the biological sample is from a biopsy. In some aspects, the biological sample is from a surgery sample. In some aspects, the body fluid is blood, urine, saliva, mucus, semen, vaginal fluid, amniotic fluid, cerebrospinal fluid, or a tissue fluid.
• the RNA primer comprises a poly T tail sequence.
• the RNA primer comprises a random sequence. See, e.g., Fig. 3.
• the method further comprises (f) separating the plurality of DNA amplicons from the plurality of cDNA amplicons after co-amplification.
• the plurality of DNA amplicons are separated from the plurality of cDNA amplicons using fragment size, biotin labels, and/or adapter sequence features.
• the method further comprises performing transcriptome quantification or isoform analysis.
• the method comprises production of cDNA.
• the method comprises production of a cDNA library.
• an amplified full-length cDNA library is used to prepare a 3’ RNA-seq library.
• RNA and RNA libraries can be separated according fragment sizes, biotin modifications or other methods, followed by preparing the pooled DNA and RNA equencing libraries according to the research purpose and sequencing instruments. In this example, the DNA and RNA libraries are first separated by fragment size.
• FIG. 2 shows exemplary input cells compatible with the disclosure.
• Input cells can be genetically engineered cells, antibody labelled cells or barcoded cells.
• FIG. 3 shows an exemplary flow diagram of a method of the disclosure including profiling DNA and total RNA using random primers for reverse transcription.
• FIG. 4 shows an exemplary flow diagram of a method of the disclosure using a single cell/nucleus suspension.
• a single cell/nucleus suspension is first used to perform an in situ reverse transcription reaction when the cell/nucleus remains intact. Then, the single cell/nucleus is dispensed into a single tube/well. Next, lysis and tagmentation are performed to fragment DNA and add DNA adaptors. Subsequently, the DNA and cDNA are coamplified in the same reaction vessel. Barcoded DNA and RNA of each single tube/well are then mixed together (shown as step 5), and DNA and RNA are separated. Finally the RNA and DNA libraries are enriched individually to prepare sequencing libraries.
• FIG. 6 shows an exemplary flow diagram of a method of the disclosure.
• the combinatorial barcodes are assigned to the same end of the DNA molecule, such as the 3 ’end.
• FIG. 7 shows an exemplary flow diagram of a method of the disclosure.
• the combinatorial barcodes (bc3 and bc4) are labelled at the 5’ ends of the RNA molecules by two rounds of PCR reactions.
• FIG. 10 shows an exemplary flow diagram of a method of the disclosure.
• cells labelled with polyA based oligonucleotides like lipid-based or antibody based sample multiplexing
• genetic enginerred cells such as CRISPR-Cas9
• WellDR- seq can be used to profile DNA, RNA and the cell labels together.
• FIG. 11 shows an exemplary flow diagram of a method of the disclosure. Using cells labelled with DNA adaptors, WellDR-seq can profile DNA, RNA and the cell labels concurrently.
• FIGs. 12A-12B show low-throughput single tube experiments of 12 cells for single cell DNA copy number profiling and RNA expression analysis.
• FIG. 12A shows copy number profiles from 12 single cells profiled for the wellDR-seq method using single tube compartments. Each row represents the copy number profile from a single cell, with Log2 segment ratios showing copy number gains (red) and losses (blue).
• FIG. 12B shows quality control metrics for RNA expression profiles from the same 12 single cells depicted in FIG. 12A.
• FIGs. 13A-13G show mid-throughput for a wellDR-seq method using 384 well plates to profile breast cancer cells (from MDA-MB-231 cell line).
• FIG. 13 A is a uniform manifold approximation and projection (UMAP) depicting two clusters identified by the single cell gene expression data.
• FIG. 13B shows the top 10 differential expressed gene between cluster 0 and 1 from the RNA expression data.
• FIG. 13C shows a UMAP of RNA profiles with annotations showing the plates from which the cells were profiled.
• FIG. 13D shows Pearson correlation of gene expression (MDA-MB-231 cell line) detected by wellDR-seq (WDR) and 3’DE-seq (Takara).
• FIG. 13E shows Pearson correlation of gene expression (MDA-MB-231 cell line) detected by wellDR-seq (WDR) 10X Genomics’ single cell 3’ RNA-seq (tenx).
• FIG. 13F shows DNA superclones mapped to the UMAP of the RNA high-dimensional space.
• FIG. 13G shows a heatmap of DNA copy number aberrations from the scDNA data, in which superclones and subclones were based on the heatmap clustering results.
• Left side bar shows the plates that each cell was sequenced from.
• RNA_Cluster side-bar shows the RNA clustering results using gene expression profiles from FIG 13 A.
• FIGs. 14A-14G show result from a high-throughput nanowell based wellDR-seq method of the MDA-MB-231 cancer cell line.
• FIG. 14A is a UMAP showing two clusters of single cells from MDA-MB-231 identified by gene expression data.
• FIG. 14B shows the top 10 differential expressed genes between cluster 0 and 1.
• FIG. 14C shows gene (nFeature RNA), UMI (nCount RNA) and mitochondrial percentages of the two RNA clusters.
• FIG. 14D shows Pearson correlation of gene expression from MDA-MB-231 detected by wellDR-seq (WDR) and 3’DE-seq (Takara).
• WDR wellDR-seq
• Takara 3’DE-seq
• wellDR-seq methods comprising simultaneously co-amplifying DNA and RNA from a single biological sample in one compartment (e.g., cell/sample/well) and does not require physically separating the nucleic acids (DNA/RNA) from low input materials or single cells prior to performing amplifications and constructing sequencing libraries for sequencing.
• a polynucleotide can be singlestranded or double-stranded and, where desired, linked to a detectable moiety.
• a polynucleotide can include hybrid molecules, e.g., comprising DNA and RNA.
• G,” “C,” “A,” “T” and “U” each generally stands for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively.
• ribonucleotide” or “nucleotide” can also refer to a modified nucleotide or a surrogate replacement moiety.
• label and “detectable label” refer to a particle, ion, isotope, small molecule, macromolecule, molecular complex, or other suitable material capable of use for detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, semiconductor nanoparticles, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like.
• fluorescer refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range.
• a first polynucleotide of interest and a second polynucleotide of interest are each tagged with combinatorial barcodes A, B, and C, arranged in the 5’ to 3’ order, but the combinatorial barcodes are attached to the 5’ end of the first polynucleotide, and are attached to the 3’ end of the second polynucleotide.
• the first and second polynucleotides of interest are uniquely identifiable despite comprising the same different sequences A, B, and C, arranged in the same order, based on the arrangement ABC relative to the first or second polynucleotide of interest.
• the cell is a live cell, a genetically engineered cell, a perturbed cell (such as using CRISPR/CAS9 to perform multi-locus gene perturbation as described in Perturb-seq (Dixit et.al, Cell, 2016)), and/or a fixed cell.
• the DNA and the RNA are from a sample of micro-dissected tissue. In some aspects, the DNA and the RNA are from a biopsy.
• Certain aspects of the disclosure are directed to a method of co-amplifying DNA and RNA from a single biological sample, the method comprising: lysing a biological sample to release a plurality of nucleic acids comprising both gDNA and RNA from the biological sample; fragmenting the DNA; and attaching a DNA adaptor to the fragmented DNA from to form a plurality of DNA fragment-adaptor molecules.
• RNA is not fragmented during the fragmenting of the DNA.
• the DNA is fragmented into shortened fragments or sections of DNA.
• an adaptor is added (e.g., ligated to) the fragmented DNA.
• the fragmenting of the DNA comprises contacting the nucleic DNA with a transposase.
• the transposase is a Tn5 transposase.
• the Tn5 transposase is EZTn5TM, NexteraV2, or TS-Tn5059.
• Selective fragmentation and tagging of DNA i.e., tagmentation
• the transposable oligonucleotide adapter comprises a common priming site for DNA-specific amplification to allow amplification of the generated DNA fragments using a set of universal DNA-specific primers.
• tagmentation and hyperactive transposases useful for carrying out the method, see, e.g., U.S. Pat. Nos. 9,080,211; 9,238,671; 6,294,385; 8,383,345; 9,040,256; 9,074,251; 7,083,980; and 8,829,171; U.S. Patent Application Publication No. 2015/0291942; and Brouilette et al. (2012) Dev. Dyn. 241(10): 1584-1590; Petzke et al.
• the oligonucleotides that attach to the transposase can have a barcode or part of a barcode sequence included for distinguishing different cells, while the oligonucleotides also serve as the identifier to distinguish the DNA from the RNA molecular in the same input material (e.g., single cell/nuclei), which are in the same compartment.
• the transposome can be inactivated or inhibited by protein denaturing detergents (e.g., SDS) or heating with EDTA or other inhibitors after the tagmentation reaction.
• the adaptors that are added to the 5' and/or 3' end of a nucleic acid can comprise a universal sequence.
• a universal sequence is a region of nucleotide sequence that is common to, i.e., shared by, two or more nucleic acid molecules.
• the two or more nucleic acid molecules also have regions of sequence differences.
• the 5' adapters can comprise identical or universal nucleic acid sequences and the 3' adapters can comprise identical or universal sequences.
• a universal sequence that may be present in different members of a plurality of nucleic acid molecules can allow the replication or amplification of multiple different sequences using a single universal primer that is complementary to the universal sequence.
• Certain aspects of the disclosure are directed to a method of co-amplifying DNA and RNA from a single biological sample, the method comprising: lysing a biological sample to release a plurality of nucleic acids comprising both DNA and RNA from the biological sample and synthesizing cDNA from the RNA, wherein the synthesizing comprises reverse transcription comprising a reverse transcriptase and an RNA primer, wherein the RNA primer comprises an RNA adaptor which is distinguishable from the DNA adaptor to form a plurality of cDNA- adaptor molecules.
• the RNA can be amplified by reverse transcribing RNA into cDNA with a reverse transcriptase, and then performing PCR (i.e., RT-PCR), as described above.
• PCR i.e., RT-PCR
• a single enzyme may be used for both steps as described in U.S. Pat. No.
• cDNA can be generated from all types of RNA, including mRNA, non-coding RNA, microRNA, siRNA, and viral RNA.
• the RNA or cDNA molecules are attached (e.g., ligated) to specific adaptors, which are distinguishable from the DNA adaptors of the disclosure.
• the RNA molecules are converted into cDNA (complimentary DNA) by reverse transcriptase (e.g., MMLV reverse transcriptase, AMV reverse transcriptase or other RT enzymes).
• reverse transcriptase e.g., MMLV reverse transcriptase, AMV reverse transcriptase or other RT enzymes.
• the primer used for reverse transcription (RT) can prime using RNA molecules with poly-T tails, or random sequences, as well as other targeted gene specific sequences.
• the RNA primer has a universal sequence component (e.g., greater or eqal to about 6 base pairs) that can be used as the RNA adaptor (e.g., PCR amplification handle) sequence for amplifying the cDNA in later steps of the method.
• the primer can comprise a cell barcode or part of a cell barcode allowing for cell identity.
• the primer can comprise a unique molecular identifier (UMI) sequence to distinguish individual transcripts from PCR duplicate reads.
• the primer can comprise modifications (such as biotin), which can be used to separate the RNA from DNA molecular afterward co-amplification.
• the cell barcode can comprise 1 or 2 oligonucleotide sequences, or multiple oligonucleotide sequences.
• a template switch oligonucleotide TSO
• TSO sequence(s) can comprise a cell barcode or part of a cell barcode, serving as a cell identifier, and/or can also have a unique molecular identifier (UMI) sequence to distinguish individual transcripts from PCR duplicate reads.
• UMI unique molecular identifier
• the TSO can comprise modifications (such as biotin) used to separate the RNA from DNA molecules after co- amplifiation.
• the sequences used to label the DNA can comprise at least two distinct sequences (e.g., oligonucleotide attached on ME sequence of the transposome; or the sequence(s) used to label the RNA (e.g., RT primers and/or TSO sequences)).
• the RNA primer comprises a poly T tail universal sequences. In some aspects, the RNA primer comprises template switching oligonucleotides. In some aspects, the barcoding comprises using unique sequence identifiers or primer biotinylation.
• the adaptors that are added to the 5' and/or 3' end of a nucleic acid can comprise a universal sequence.
• a universal sequence is a region of nucleotide sequence that is common to, i.e., shared by, two or more nucleic acid molecules.
• the two or more nucleic acid molecules also have regions of sequence differences.
• the 5' adapters can comprise identical or universal nucleic acid sequences and the 3' adapters can comprise identical or universal sequences.
• a universal sequence that may be present in different members of a plurality of nucleic acid molecules can allow the replication or amplification of multiple different sequences using a single universal primer that is complementary to the universal sequence.
• the separated DNA and/or RNA amplification products can be used to prepare different sequencing libraries, e.g., according to the research purpose and sequencing instrument requirements.
• the separated DNA and/or RNA amplification products can also be used for further enrichment using DNA or RNA specific adaptors added during the previous steps.
• further enriched products can be used as input materials for performing high throughput sequencing platforms if the PCR primers used to amplify the DNA modality are the same sequencing adaptor used for the sequencing reactions.
• these methods can be modified to prepare different sequencing libraries (e.g., according to the research purpose and sequencing instrument requirements).
• high throughput sequencing can be performed.
• the high throughput sequencing platforms include, but are not limited to, next generation sequencing, single molecule sequencing and nanopore sequencing.
• the amplified DNA can be used for profiling copy number variations/alterations (CNV/CNA), structure variations (SVs), indels and point mutations.
• the amplified DNA can be used for profiling targeted genes or gene panels, probebased target capture, exon capture or other capture applications Fig 9.
• the amplified DNA can be used for investigating DNA rearrangements and markers, detecting different frequency of mutations, profiling epigenetics modifications, DNA and protein interactions, and other DNA related applications.
• the sequencing comprises paired-end sequencing or single-read sequencing.
• the sequencing comprises next-generation sequencing (NGS).
• NGS next-generation sequencing
• the amplified DNA or cDNA library can be sequenced and analyzed using methods known to those of skill in the art, e.g., by next-generation sequencing (NGS).
• NGS next-generation sequencing
• RNA expression profiles are determined using any sequencing methods known in the art. Determination of the sequence of a nucleic acid sequence of interest can be performed using a variety of sequencing methods known in the art including, but not limited to, sequencing by synthesis (SBS), sequencing by hybridization (SBH), sequencing by ligation (SBL) (Shendure et al.
• High-throughput sequencing methods e.g., using platforms such as Roche 454, Illumina Solexa, AB-SOLiD, Helicos, Complete Genomics, Polonator platforms and the like, can also be utilized.
• platforms such as Roche 454, Illumina Solexa, AB-SOLiD, Helicos, Complete Genomics, Polonator platforms and the like.
• a variety of light-based sequencing technologies are known in the art (Landegren et al. (1998) Genome Res. 8:769-76; Kwok (2000) Pharmacogenomics 1 :95-100; and Shi (2001) Clin. Chem. 47: 164-172).
• the method further comprises identifying a mutation in the DNA or RNA.
• the expression profiling methods described herein are also useful for ascertaining the effect of the expression of one or more nucleic acid sequences (e.g., genes, mRNAs and the like) on the expression of other nucleic acid sequences (e.g., genes, mRNAs and the like) in the same cell or in different cells. This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.
• nucleic acid sequences e.g., genes, mRNAs and the like
• the expression profiling methods described herein are also useful for ascertaining differential expression patterns of one or more nucleic acid sequences (e.g., genes, mRNAs and the like) in normal and abnormal cells. This provides a battery of nucleic acid sequences (e.g., genes, mRNAs and the like) that could serve as a molecular target for diagnosis or therapeutic intervention.
• nucleic acid sequences e.g., genes, mRNAs and the like
• the mutation is a single nucleotide variation.
• the mutation is associated with a phenotype of interest.
• the method further comprises performing transcriptome quantification or isoform analysis.
• the dislosed methods can be used for low-, middle- and high throughput whole genome (DNA) and transcriptome (RNA) co-amplification and library preparation.
• the disclosed methods use two different sets of adaptors to barcode DNA and RNA separately (one set for DNA, one set for RNA) from the same single cells or input materials.
• the approach uses different barcodes combinations to assign cell barcodes for each modality (DNA or RNA) of each cell, and then pools all of the barcoded amplified product together to prepare DNA and RNA sequencing libraries separately.
• the disclosed method is a flexible whole genome and transcriptome co-amplification method, which can add different adaptor sets to label and amplify DNA and RNA from the same input materials/single cell simultaneously.
• the disclosed methods assign sample/cell barcodes to DNA and RNA modalities from different input materials/single cells by a nested multiplexing PCR to index DNA & RNA simultaneously using different barcode combinations.
• the disclosed methods can amplify whole genomes and whole transcriptomes simultaneously from single cells, or tens to millions of cells, or alternatively from from extracted or unextracted DNA/RNA materials or limited materials.
• the disclosed methods enables preparing DNA sequencing libraries from the input materials/cells together after pooling, and also enables preparing the RNA sequencing libraries from all input materials/cells together after pooling, which avoids preparing DNA and/or RNA sequencing libraries individually from each cell one- by-one.
• this feature allows for the claimed methods (e.g., wellDR-seq) to be highly scalable for both tubes or plates formats, as well as very high-throughput platforms such as nanowells or nanochips.
• Certain aspects of the disclosure comprise the addition of adaptors to DNA fragments and RNA/cDNA in a mixed solution of the nucleic acids and does not require physical separation of the DNA and RNA nucleic acids during addition of the adaptors. This is in contrast to other methods of creating genomic and transcriptomic libraries from a single souce like G&T- seq (Macaulay et al., Nat Methods 12, 519-522 (2015)), SIDR-seq (Han et al., SIDR: Genome Research 28, 75-87 (2016)), and DNTR-seq (Zachariadis et al., Molecular Cell 80, , 541-553. e5 (2020)).
• the certain methods disclosed herein have other advantages over DR-Seq including that the present methods can be used for high-throughput analysis.
• the method of DR-Seq first entails reverse transcription, then uses quaslinear amplification to amplify DNA and RNA. Because of this quaslinear amplification strategy, DR-Seq cannot achieve high throughput cell barcoding. Further, the DNA and RNA library of each single cell needs to be prepared separately in DR-Seq, which requires effort and cost to prepare (Dey, et al., Nat Biotechnol 33, 285-289 (2015)).
• Certan aspects of the present disclosure are different from the scONE-seq method (Wu, et al., (2021)).
• scONE-seq uses the same adaptors to label DNA and RNA, which does not allow the separation of DNA and RNA molecules during library preparation. This also does not allow users to control the sequencing depth for the DNA and RNA assays, which need to be sequenced at different depths.
• certain methods disclosed herein use different adaptor combinations to distinctly label DNA and RNA.
• the assays (DNA or RNA) can then be enriched during the preamplification step, post-amplification step, and also after merging all of the libraries from all cells.
• the fragment size of DNA and RNA assay can also distinguish DNA and RNA.
• the methods of the disclosure e.g., wellDR-seq
• the methods of the disclosure comprise labelling the adaptors or primers of either assay (DNA or RNA) with base modifications (eg. biotin) to further separate DNA and RNA assays after co-amplification.
• certain methods of the present disclosure uses combinatorial barcodes for both DNA and RNA assays, which can enable profiling the genome and transcriptome from hundreds or thousands of cells or low input materials.
• the methods disclosed herein e.g., wellDR-seq
• the present methods use the substrate specificity of the transposase and RNA ligase enzymes to selectively attach DNA-specific adapters to DNA and RNA-specific adapters to RNA, respectively in the pooled mixtures.
• kits for performing the method of claim 1 comprising: a) a Class 2 transposase; b) a transposable oligonucleotide comprising an oligonucleotide adapter comprising a common priming site for DNA-specific amplification; c) a 5' oligonucleotide adapter comprising a 5' common priming site for RNA- specific amplification; d) a 3' oligonucleotide adapter comprising a 3' common priming site for RNA-specific amplification; k) an RNase inhibitor; 1) a reverse transcriptase; m) a DNA polymerase; n) a set of DNA indexing PCR primers; and o) a set of RNA indexing PCR primers.
• the kit further comprises reagents for performing next-generation sequencing.
• oligonucleotide adapters e.g., adapter comprising a common priming site for DNA-specific amplification, a 5' adapter comprising a 5' common priming site for RNA-specific amplification, a 3' oligonucleotide adapter comprising a 3' common priming site for RNA-specific amplification
• RNase inhibitor e.g., RNase inhibitor
• reverse transcriptase e.g., DNA polymerase (e.g., Taq polymerase for PCR), DNA indexing PCR primers
• RNA indexing PCR primers can be provided in kits with suitable instructions and other necessary reagents in order to carry out preparation of RNA and DNA sequencing libraries as described above.
• the kit will contain in separate containers the various primers, adapters, and enzymes, and other reagents required to carry out the method.
• instructions e.g., written, CD-ROM, DVD, flash drive, SD card, digital download etc.
• RNA and DNA sequencing libraries simultaneously as described herein will be included with the kit.
• the kit may also contain other packaged reagents and materials (e.g., wash buffers, nucleotides, silica spin columns, capture probes for ribosomal RNA depletion, and other reagents and/or devices for performing e.g., clonal amplification, digital PCR, NGS sequencing, ribosomal RNA depletion, nucleic acid purification, and the like).
• other packaged reagents and materials e.g., wash buffers, nucleotides, silica spin columns, capture probes for ribosomal RNA depletion, and other reagents and/or devices for performing e.g., clonal amplification, digital PCR, NGS sequencing, ribosomal RNA depletion, nucleic acid purification, and the like).
• RNA adaptor PCR priming
• other methods can be used to add the RNA adaptor (PCR priming) sequences to the second end of the cDNA molecules, such as performing a ligation reaction at the 3 ’end of the cDNA, or performing a second strand synthesis using oligonucleotides with random priming sequences and universal tails, or oligonucleotides with target priming sequence and universal tails.
• the RNA adaptors (PCR priming) sequences could have modifications (such as biotin) that will be used to separate the RNA from DNA molecules after the initial amplification reactions.
• the primers can have a cell barcode or part of a cell barcode serving as a cell identity, the primers can also have modifications (such as biotin) that are used to separate the pool of RNA from DNA molecules afterward.
• the primer pairs for the DNA assay and pairs for the RNA assay are all added into the same reaction during exponential coamplification.
• the primers can have cell barcodes or part of cell barcodes serving as the cell identity, and the primers can also have modifications (such as biotin) used to separate the RNA from DNA molecules afterwards.
• the annealing temperatures of the co-amplification step can be used to favor one of the assays or both assays to control the total number of molecules amplified. By controlling the favored annealin temperature, the DNA and RNA assay product amounts can be balanced in alternative manner.
• the DNA or RNA from the same cell/sample are labelled with cell/sample barcodes.
• the DNA and RNA from the same cell/sample material can be labelled with same barcode sets or different barcode sets (by knowing the DNA/RNA barcode correspondence relationship).
• the DNA and RNA modalities can be barcoded by single or multiple barcodes, or the combination of barcodes.
• the two barcodes, three barcodes or multiple barcodes (>3) can be located in one end (eg 5’) of each modality, or alternatively can be located in both ends (eg 5’ and 3’) of each modality.
• the wellDR-seq method can use tagmentation based chemistries (eg. Tn5 transposome) to fragment DNA.
• the DNA fragments can be barcoded by using tagmentation enzyme with different adaptors (eg. by attaching different oligonucleotide sequences to the mosaic sequences of Tn5 transposase), or can be barcoded in the subsequent PCR steps through PCR primers that contain different barcodes (indices) or barcode combinations, or barcoded by both approaches by combining the barcode introduced in tagmentation step and PCR steps.
• cDNA pre-amplification PCR was cycled as follows: 72 °C for 10 min, 98 °C for 3 min, 6 cycles of 98 °C for 20s, 55 °C for 30s, 72°C for 150s.
• the amplified products were pooled into one tube.
• the pooled sample was first double-selected by 0.6X-1.8X Ampure beads (Beckman) purification to separate DNA final library and full-length cDNA.
• the purified full-length cDNA was further purified by 0.8X Ampure beads purification.
• wellDR-seq was used to profile the genome and transcriptome of single cells from the MDA- MB-231 cell line using 384-well plates.
• the wellDR-seq libraries were prepaed from three 384-well plates. In each plate, single cells were sorted into 380 wells, 10 cells to two wells as positive control and zero cell to the other two wells as negative controls.
• 80M reads were sequenced in total (20- 50k reads/cell), of which 62% mapped to the transcriptome regions. A total of 767 (67%) single cells passed QC. On average, 21,641 UMI and 3,317 genes were detected in each single cell.
• the wellDR-seq method was applied to amplify and prepare DNA and RNA libraries simultaneously from thousands of single cells in parallel from the MDA-MB-231 breast cancer cell line by using nanowell chips to demonstrate a high-throughput application.
• Single cell suspensions were dispensed into 5184-wells nanowell chips (ICELL8), and selected 1763 single cells to perform the wellDR-seq protocol.
• ICELL8 5184-wells nanowell chips
• 98M reads were sequenced with the correct wellDR-seq RNA library structure, of which 80% of the reads mapped to the transcriptome.
• Example 9 Performing wellDR-seq in single tubes/ 96-well plates with RNA-first labeling chemistry
• Reverse transcription was carried out at 50 °C for 60 min, and the reaction was stopped by incubating at 80 °C for 10 min.
• 6ul of tagmentation mix containing 2X TD buffer (in-house), 20mM MgC12 and 0.2 ul TDE1 (Illumina) were added to each tube/well.
• Tagmentation reaction was carried out at 55 °C for 10 min.
• 2 ul of neutralization mix containing 210mM EDTA were added to each tube/well.
• Neutralization was carried out at 50 °C for 30 mins.
• two PCR programs were tested for the RNA first version of wellDR-seq in single tubes/well. The first PCR program is using 36ul PCR mix buffer.
• RNA_N7XX primers [AAGCAGTGGTATCAACGCAGAGTAC-N8(8bp, RNA Cell Barcodel)- NNNNNNNNNNGAGGCGTAGTGGCT (SEQ ID NO: 3)]
• DNA N7XX primers [CAAGCAGAAGACGGCATACGAGAT-N8(8bp, DNA_Cell_Barcode2)- GTCTCGTGGGCTCGG (SEQ ID NO: 4)]
• DNA S5XX primers [AATGATACGGCGACCACCACCGAGATCTACAC-N8(8bp, DNA Cell Barcodel)- TCGTCGGCAGCGTC (SEQ ID NO: 5)] were added into each well/tube.
• Co-amplification PCR of DNA and cDNA was cycled as follows: 22 cycles of 98 °C for 20s, 60 °C for 30s, 72°C for 90s, then 72 °C for 5 min.
• the second PCR program is using 8ul PCR program.
• RNA_N7XX primers [AAGCAGTGGTATCAACGCAGAGTAC-N8(8bp, RNA Cell Barcodel)- NNNNNNNNGAGGCGTAGTGGCT (SEQ ID NO: 3)].
• Pre-amplification PCR of cDNA was cycled as follows: 8 cycles of 98 °C for 20s, 60 °C for 30s, 72°C for 90s were added into each well/tube, then 72 °C for 5 min. Next, 2ul of PCR mix2 containing 0.5X KAPA HiFi GC Buffer, 0.13 uM of DNA_N7XX primers [CAAGCAGAAGACGGCATACGAGAT-N8(8bp, DNA_Cell_Barcode2)-GTCTCGTGGGCTCGG (SEQ ID NO: 4)], and 0.13 uM of DNA S5XX primers [AATGATACGGCGACCACCACCGAGATCTACAC-N8(8bp, DNA Cell Barcodel)- TCGTCGGCAGCGTC (SEQ ID NO: 5)] were added into each well/tube.
• Co-amplification PCR of DNA and cDNA was cycled as follows: 22 cycles of 98 °C for 20s, 60 °C for 30s, 72°C for 90s, then 72 °C for 5 min. The protocol as outlined in Example 5 was then followed to finish the final preparation of the DNA and RNA libraries.

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