NZ749719B2 - Single cell whole genome libraries and combinatorial indexing methods of making thereof - Google Patents
Single cell whole genome libraries and combinatorial indexing methods of making thereof Download PDFInfo
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
Abstract
Provided herein are methods for preparing a sequencing library that includes nucleic acids from a plurality of single cells. In one embodiment, the sequencing library includes whole genome nucleic acids from the plurality of single cells. In one embodiment, the method includes generating nucleosome-depleted nuclei by chemical treatment while maintaining integrity of the nuclei. Also provided herein are compositions, such as compositions that include chemically treated nucleosome-depleted isolated nuclei. depleted nuclei by chemical treatment while maintaining integrity of the nuclei. Also provided herein are compositions, such as compositions that include chemically treated nucleosome-depleted isolated nuclei.
Description
SINGLE CELL WHOLE GENOME LIBRARIES AND COMBINATORIAL INDEXING
METHODS OF MAKING THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application Serial No.
62/365,916, filed July 22, 2016, and U.S. Provisional Application Serial No. 62/451,305, filed
January 27, 2017, each of which are incorporated by reference herein.
SEQUENCE LISTING
This application contains a Sequence Listing electronically submitted via EFS-Web to the
United States Patent and Trademark Office as an ASCII text file entitled
"1592SeqListing ST25.txt" having a size of 27 kilobytes and created on July 18, 2017. The
information contained in the Sequence Listing is incorporated by reference herein.
FIELD
Embodiments of the present disclosure relate to sequencing nucleic acids. In particular,
embodiments of the methods and compositions provided herein relate to producing indexed
single-cell sequencing libraries and obtaining sequence data therefrom.
BACKGROUND
Single cell sequencing has uncovered the breadth of genomic heterogeneity between cells
in a variety of contexts, including somatic aneuploidy in the mammalian brain (McConnell, M. J.
et al. Science (80.). 342, 632-637 (2013), Cai, X. et al. Cell Rep. 8, 1280-1289 (2014), Knouse,
K. A. et al., Proc Natl Acad Sci U S A 111, 13409-13414 (2014), Rehen, S. K. et al. Proc. Natl.
Acad. Sci. U. S. A. 98, 13361-6 (2001)) and intra-tumor heterogeneity (Navin, N. et al. Nature
472, 90-94 (2011), Eirew, P. et al. Nature 518, 422-6 (2014), Gawad, C. et al. Proc. Natl. Acad.
Sci. U. S. A. 111, 17947-52 (2014), Gao, R. et al. Nat. Genet. 1-15 (2016).
doi:10.1038/ng.3641). Studies have taken one of two approaches: high depth of sequencing per
cell for single nucleotide variant detection (Cai, X. et al. Cell Rep. 8, 1280-1289 (2014), Zong,
C. et al. Science (80-. ). 338, 1622-1626 (2012)), or low-pass sequencing to identify copy
number variants (CNVs) and aneuploidy (McConnell, M. J. et al. Science (80.). 342, 632-637
(2013), Baslan, T. et al. Genome Res. 125, 714-724 (2015), Knouse, K. A. et al. Genome Res.
gr.198937.115- (2016). doi:10.1101/gr.198937.115). In the latter approach, the lack of an
efficient, cost-effective method to produce large numbers of single cell libraries has made it
difficult to quantify the frequency of CNV-harboring cells at population scale, or to provide a
robust analysis of heterogeneity in the context of cancer (Gawad, C. et al. Nat. Rev. Genet. 17,
175-88 (2016)).
Recently, contiguity-preserving transposition (CPT-seq) was established, a method to
produce thousands of individually barcoded libraries of linked sequence reads using a
transposase-based combinatorial indexing strategy (Adey, A. et al. Genome Biol. 11, R119
(2010), Amini, S. et al. Nat. Genet. 46, 1343-9 (2014), Adey, A. et al. Genome Res. 24, 2041-
2049 (2014)). We applied CPT-seq to the problem of genomic haplotype resolution (Amini, S. et
al. Nat. Genet. 46, 1343-9 (2014)) and de novo genome assembly (Adey, A. et al. Genome Res.
24, 2041-2049 (2014)). This concept was then integrated into the chromatin accessibility assay,
ATAC-seq (Buenrostro, J. D. et al. Nat. Methods 10, 1213-8 (2013)), to produce profiles of
active regulatory elements in thousands of single cells (Cusanovich, D. a et al. Science 348, 910-
4 (2015)) (sciATAC-seq, ). In combinatorial indexing, nuclei are first barcoded by the
incorporation of one of 96 indexed sequencing adaptors via transposase. The 96 reactions are
then combined and 15-25 of these randomly indexed nuclei are deposited into each well of a
PCR plate by Fluorescence Activated Nuclei Sorting (FANS, . The probability of any two
nuclei having the same transposase barcode is therefore low (6-11%)(Cusanovich, D. a et al.
Science 348, 910-4 (2015)). Each PCR well is then uniquely barcoded using indexed primers. At
the end of this process, each sequence read contains two indexes: Index 1 from the transposase
plate, and Index 2 from the PCR plate, which facilitate single cell discrimination. As proof of
principle, Cusanovich and colleagues produced over 15,000 sciATAC-seq profiles and used
them to separate a mix of two cell types by their accessible chromatin landscapes (Cusanovich,
D. a et al. Science 348, 910-4 (2015)).
Although high cell count single-cell sequencing has shown its efficacy in separation of
populations within complex tissues via transcriptomes, chromatin-accessibility, and mutational
differences, it has not been possible until now to obtain sequence information that includes the
whole genome of single cells.
SUMMARY OF THE APPLICATION
Provided herein are methods for preparing a sequencing library that includes nucleic
acids from a plurality of single cells. In one embodiment, the method includes providing isolated
nuclei from a plurality of cells; subjecting the isolated nuclei to a chemical treatment to generate
nucleosome-depleted nuclei while maintaining integrity of the isolated nuclei; distributing
subsets of the nucleosome-depleted nuclei into a first plurality of compartments and contacting
each subset with a transposome complex, where the transposome complex in each compartment
includes a transposase and a first index sequence that is different from first index sequences in
the other compartments; fragmenting nucleic acids in the subsets of nucleosome-depleted nuclei
into a plurality of nucleic acid fragments and incorporating the first index sequences into at least
one strand of the nucleic acid fragments to generate indexed nuclei that include indexed nucleic
acid fragments, where the indexed nucleic acid fragments remain attached to the transposases;
combining the indexed nuclei to generate pooled indexed nuclei; distributing subsets of the
pooled indexed nuclei into a second plurality of compartments; incorporating into the indexed
nucleic acid fragments in each compartment a second index sequence to generate dual-index
fragments, where the second index sequence in each compartment is different from second index
sequences in the other compartments; and combining the dual-index fragments, thereby
producing a sequencing library that includes whole genome nucleic acids from the plurality of
single cells.
In one embodiment, the chemical treatment includes a treatment with a chaotropic agent
capable of disrupting nucleic acid-protein interactions, such as lithium 3,5-diiodosalicylic acid.
In one embodiment, the chemical treatment includes a treatment with a detergent capable of
disrupting nucleic acid-protein interactions, such as sodium dodecyl sulfate (SD S).
In one embodiment, the nuclei are treated with a cross-linking agent before subjecting the
isolated nuclei to the chemical treatment, such as formaldehyde. The cross-linking agent can be
at a concentration from about 0.2% to about 2%, and in one embodiment is about 1.5%. In one
embodiment, the cross-linking by formaldehyde is reversed after distributing subsets of the
pooled indexed nuclei and before incorporating into the indexed nucleic acid fragments in each
compartment a second index sequence. In one embodiment, the reversal of the cross-linking
includes incubation at about 55°C to about 72°C. In one embodiment, the transposases are
disassociated from the indexed nucleic acid fragments prior to the reversal of the cross-linking.
In one embodiment, the transposases are disassociated from the indexed nucleic acid fragments
using sodium dodecyl sulfate (SDS).
In one embodiment, the nuclei are treated with a restriction enzyme prior to fragmenting
nucleic acids in the subsets of nucleosome-depleted nuclei into a plurality of nucleic acid
fragments and incorporating the first index sequences. In one embodiment, the nuclei are treated
with a ligase after treatment with the restriction enzyme.
In one embodiment, the distributing subsets of the nucleosome-depleted nuclei, the
distributing subsets of the pooled indexed nuclei, or the combination thereof, is performed by
fluorescence-activated nuclei sorting. In one embodiment, the subsets of the nucleosome-
depleted nuclei include approximately equal numbers of nuclei, and in one embodiment, the
subsets of the nucleosome-depleted nuclei include from 1 to about 2000 nuclei. In one
embodiment, the subsets of the pooled indexed nuclei include approximately equal numbers of
nuclei, and in one embodiment, the subsets of the pooled indexed nuclei include from 1 to about
nuclei. In one embodiment, the subsets of the pooled indexed nuclei include at least 10 times
fewer nuclei than the subsets of the nucleosome-depleted nuclei, or at least 100 times fewer
nuclei than the subsets of the nucleosome-depleted nuclei.
In one embodiment, the first plurality of compartments, the second plurality of
compartments, or the combination thereof, is a multi-well plate, such as a 96-well plate or a 384 -
well plate.
In one embodiment, the transposome complex is added to the compartments after the
subsets of nucleosome-depleted nuclei are distributed into the compartments. In one
embodiment, each of the transposome complexes includes a transposon, and each of the
transposons includes a transferred strand. In one embodiment, the transferred strand includes the
first index sequence and a first universal sequence.
In one embodiment, the incorporation of the second index sequence into the indexed
nucleic acid fragments includes contacting the indexed nucleic acid fragments in each
compartment with a first universal primer and a second universal primer, each including an index
sequence and each including a sequence identical to or complementary to a portion of the first
universal sequence, and performing an exponential amplification reaction. In one embodiment,
the exponential amplification reaction can be a polymerase chain reaction (PCR), and in one
embodiment, the PCR can include 15 to 30 cycles. In one embodiment, the index sequence of
the first universal primer is the reverse complement of the index sequence of the second
universal primer, and in another embodiment, the index sequence of the first universal primer is
different from the reverse complement of the index sequence of the second universal primer. In
one embodiment, the first universal primer further includes a first capture sequence and a first
anchor sequence complementary to a universal sequence at the 3' end of the dual-index
fragments, and in one embodiment, the first capture sequence includes the P5 primer sequence.
In one embodiment, the second universal primer further includes a second capture sequence and
a second anchor sequence complementary to a universal sequence at the 5' end of the dual-index
fragments, and in one embodiment, the second capture sequence includes the reverse
complement of the P7 primer sequence.
The method can also include an enrichment of dual-index fragments using a plurality of
capture oligonucleotides having specificity for the dual-index fragments. In one embodiment,
the capture oligonucleotides are immobilized on a surface of a solid substrate, and in one
embodiment, the capture oligonucleotides include a first member of a universal binding pair and
a second member of the binding pair is immobilized on a surface of a solid substrate.
The method can also include sequencing of the dual-index fragments to determine the
nucleotide sequence of nucleic acids from the plurality of single cells. In one embodiment, the
method can include providing a surface that includes a plurality of amplification sites, where the
amplification sites include at least two populations of attached single stranded capture
oligonucleotides having a free 3' end, and contacting the surface that includes amplification sites
with the dual-index fragments under conditions suitable to produce a plurality of amplification
sites that each include a clonal population of amplicons from an individual dual-index fragment.
In one embodiment, the number of the dual-index fragments exceeds the number of amplification
sites, where the dual-index fragments have fluidic access to the amplification sites, and where
each of the amplification sites includes a capacity for several dual-index fragments in the
sequencing library. In one embodiment, the contacting includes simultaneously (i) transporting
the dual-index fragments to the amplification sites at an average transport rate, and (ii)
amplifying the dual-index fragments that are at the amplification sites at an average
amplification rate, where the average amplification rate exceeds the average transport rate.
Also provided herein are compositions. In one embodiment, a composition includes
chemically treated nucleosome-depleted isolated nuclei, where the isolated nuclei include
indexed nucleic acid fragments. In one embodiment, the isolated nuclei include non-natural
cross-links. In one embodiment, the composition includes indexed nucleic acid fragments that
terminate in a cleaved restriction site including an overhang. In one embodiment, the isolated
nuclei include rearranged genomic DNA. In another embodiment, a composition includes a
multi-well plate, where a well of the multi-well plate includes chemically treated nucleosome-
depleted isolated nuclei, where the isolated nuclei include indexed nucleic acid fragments.
BRIEF DESCRIPTION OF THE FIGURES
The following detailed description of illustrative embodiments of the present disclosure
may be best understood when read in conjunction with the following drawings.
shows a general block diagram of a general illustrative method for single-cell
combinatorial indexing according to the present disclosure.
shows a schematic drawing of an illustrative embodiment of an indexed nucleic
acid fragment.
shows a schematic drawing of an illustrative embodiment of a dual-index
fragment.
shows single cell combinatorial indexing with nucleosome depletion. ()
Single cell combinatorial indexing workflow. () Phase contrast images of intact nuclei
generated by standard isolation followed by nucleosome depletion using Lithium Assisted
Nucleosome Depletion (LAND) or crosslinking and SDS treatment (xSDS). Scale bar: 100 p.m.
() Nucleosome depletion produces genome-wide uniform coverage that is not restricted
to sites of chromatin accessibility.
shows Fluorescence Activated Nuclei Sorting (FANS). Representative plots from
FANS sorting of single nuclei. All plots are from sorting the second (PCR) plate unless noted
otherwise. () ATAC-seq Nuclei () LAND () He La S3 and 3T3 ()
xSDS () PDAC Sort 1 Transposase Plate () PDAC Sort 2 PCR plate.
shows SCI-seq single cell determination using a mixed model. HeLa.LAND3
shown. normalmixEM of the R package mixtools was used to identify each distribution: noise
index combinations (left peak) and single cell libraries (right peak). The read count threshold to
consider an index combination as a single cell library is the greater of either one standard
deviation (in log10 space) below the mean of the single cell distribution, or 2 greater (in log10
space, thus 100 fold greater) than the mean of the noise distribution and at a minimum of 1,000.
For the library shown, one standard deviation below the mean of the single cell component is
greater and therefore used as the read count threshold.
shows comparison of LAND and xSDS nucleosome depletion methods with SCI-
seq. () Complexity for one of six LAND SCI-seq preparations on GM12878. Right,
histogram showing distribution of read counts. Dashed line represents single-cell read cutoff.
() As in but for xSDS nucleosome depletion for one of three PCR plates. () Left, model built on down-sampled reads for the GM12878 xSDS preparation and used to
predict the full depth of coverage. Right, projections for one of the LAND preparations and the
full xSDS preparation. Shading represents s.d. over multiple models. Points represent actual
depth of sequencing. () Coverage uniformity scores for SCI-seq using LAND or xSDS
and for quasi-random priming (QRP) and degenerate oligonucleotide PCR (DOP). ()
Summary of the percentage of cells showing aneuploidy at the chromosome-arm level across all
preparations with and without the imposition of a variance filter. () Karyotyping results of
50 GMI2878 cells. (, ) Summary of windowed copy-number calls and clustering
of single GM 12878 cells produced using LAND (FIG, 7g) or xSDS (). In each panel top
represents a chromosome-arm-scale summary of gain or loss frequency for all cells; bottom is
the clustered profile for cells that contain at least one CND` call.
shows SCI-seq library complexity and index read count distributions for all
preparations. For each preparation two plots are shown. Left: each point represents a unique
index combination, x-axis is the fraction of unique reads assigned to that index combination, y-
axis is the log10 unique read count for the index combination. Contour lines represent point
density. Right: A histogram of the log10 unique read counts for each of the index combinations.
We expect the majority of potential index combinations not to represent a single cell library and
therefore containing very few unique reads (leftmost distribution), with the single cell libraries
having far greater read counts (right distribution, or tail in lower performance libraries). Since
the plot is on a log10 scale, the noise distribution actually only takes up a minority of the total
read counts.
shows SCI-seq on a mix of human and mouse cells. For all panels the number of
reads for each index component are plotted based on the count aligning to the human reference
genome, or the mouse reference genome. (,b) LAND nucleosome depletion on Human
(GM12878) and Mouse (3T3), (,d) LAND nucleosome depletion on Human (HeLa S3)
and Mouse (3T3), () xSDS nucleosome depletion on Human (HeLa S3) and Mouse
(3T3).
shows SCI-seq library complexity and index read count distributions after deeper
sequencing. For each preparation two plots are shown as in S2 the left plot shows fraction of
unique reads versus unique read count for each index combination. While the right plot shows a
histogram of read counts for each index combination. Cells from wells sequenced more deeply
are shown along with the rest of the plate that those wells belong to. The population of cells with
lower complexity (more to the left) is the population that has been sequenced more deeply.
shows 9bp read overlaps observed from sequencing adjacent transposition events
in the same single cell. (a) Diagram of how the 9bp copying occurs from the
transposition event. (b) Representative single cells showing the size of all amplicon
overlaps with a dashed line at 9bp.
shows copy number calling computational workflow for HMM and CBS. After
calling, call sets for CBS and HMM were intersected together with Ginkgo and only calls present
in all three sets were retained as the final call set.
shows CNV assessment using standard methods of single cell sequencing on
GM12878. Top: Summary of chromosome arm amplifications and deletions, Bottom:
hierarchical clustering of cells.
shows variance by window size and read count cutoff across all methods. Plots
showing the change in MAD or MAPD score as a function of window size and read counts per
cell.
shows GM12878 aneuploidy rates across variance score cutoffs. Each point is
the aneuploidy rate for the population of cells (y-axis), scaled by the number of cells included at
a given score cutoff (x-axis).
shows CNV profiles for Rhesus frontal cortex, Individual 1 using quasi-random
priming (QRP). (a) Ginkgo Calls, (b) CBS calls, (c) MINI calls, (d) Intersection of all three, and (e) Intersection of just CBS and MINI.
shows CNV profiles for Rhesus frontal cortex, Individual 1 using degenerate
oligonucleotide primed PCR (DOP). (a) Ginkgo Calls, (b) CBS calls, (c)
HMM calls, (d) Intersection of all three, and (e) Intersection of just CBS and
HMM.
shows CNV profiles for Rhesus frontal cortex, Individual 1 using SCI-seq with
LAND nucleosome depletion. (a) Ginkgo Calls, (b) CBS calls, (c) HMM
calls, (d) Intersection of all three, and (e) Intersection of just CBS and HMM.
shows CNV profiles for Rhesus frontal cortex, Individual 1 using SCI-seq with
xSDS nucleosome depletion. (a) Ginkgo Calls, (b) CBS calls, (c) HMM
calls, (d) Intersection of all three, and (e) Intersection of just CBS and HMM.
shows somatic CNVs in the rhesus brain. (a) Three single-cell examples
showing copy number variants, and one representative euploid cell for the SCI-seq preparation
(IIMM). (b) Frequency of aneuploidy as determined by each of the methods with and
without filtering.
shows comparison of coverage uniformity for Rhesus frontal cortex individual 1.
Uniformity measures are very similar to those of GM12878 preparations (Fig. 7b).
shows Rhesus aneuploidy rates across variance score cutoffs. Each point is the
aneuploidy rate for the population of cells (y-axis), scaled by the number of cells included at a
given score cutoff (x-axis).
shows CNV profiles for Rhesus frontal cortex, Individual 2 using SCI-seq with
xSDS nucleosome depletion. (a) Ginkgo Calls, (b) CBS calls, (c) HMM
calls, (d) Intersection of all three, and (e) Intersection of just CBS and HMM.
shows SCI-seq analysis of a stage III human Pancreatic Ductal A den ocarci noma
(PDAC). (a) SCI-seq library complexity. Right panel, histogram showing distribution of
read counts. Dashed line represents single cell read cutoff. (FIG, 24b) Breakpoint calls (top) and
breakpoint window matrix of log2 sequence depth ratio. (c) Principle component analysis
and k-means clustering on breakpoint matrix. (d) 100 kbp resolution CNV calling on
aggregated cells from each cluster. (e) Cluster specific CNVs and CEBPA amplification
present in all clusters (k4 shown).
shows SCI-seq using xSDS-based nucleosome depletion on pancreatic ductal
adenocarcinoma. Copy number call summary for 2.5 Mbp windows for the three methods of
copy number calling used in the analysis: (a) Ginkgo, (b) CBS, and (c)
HMM.
shows single cell CNV calls on primary PDAC using xSDS SCI-seq.
Representative single cell signal plots.
shows schematic of breakpoint analysis workflow. First, individual cells are
analyzed for breakpoints. Breakpoints from all cells are merged and locally summed when above
threshold. Intervals are defined between local shared breakpoints and average ratio scores are
found within each interval.
shows SCI-seq using LAND-based nucleosome depletion on HeLavS3 using the
Hidden Markov Model method for copy number variant calling. Summary of windowed (2.5
Mbp) calls and hierarchical clustering of cells. CBC copy number calling resulted in a heavy bias
against sub-chromosomal calls and Ginkgo failed to properly identify the ploidy in a number of
cells resulting in a majority of cells called as entirely amplified.
shows SCI-seq using LAND-based nucleosome depletion on He La S3 copy
number variant calling in single cells using the Hidden Markov Model method. Representative
single cell signal plots. A signal of 1 corresponds to the mean ploidy of 2.98.
shows breakpoint analysis of HeLa. (a) Breakpoints identified in the
HeLa cell line from an HMM analysis using 2.5 Mbp windows. (b) Log2 matrix of HeLa
breakpoint windows for cells normalized to GM12878.
shows PCA on HeLa breakpoint windows. HeLa produces a single population as
expected based on the stability of the cell line. Red and blue points indicate different
preparations.
shows SCI-seq using xSDS-based nucleosome depletion on a banked stage II
rectal cancer sample. Intersected copy number call summary for 2.5 Mbp windows.
shows the gating scheme used to isolate single nuclei after treatment with
transposase using forward scatter, side scatter, and DAPI intensity parameters.
shows a general block diagram of one embodiment of a general illustrative
method for single-cell combinatorial indexing and genome and chromosome conformation
according to the present disclosure.
shows the library complexity and unique read counts obtained from the method
using various formaldehyde concentrations and time of crosslink reversal.
shows an example of a single cell library using sci-GCC on HeLa. Signal
produced from chimeric ligation junction reads is shown between distal regions of the genome
over 10 Mbp windows with the first window on the x-axis and linked window on the y-axis.
Highlighted is a known translocation present in HeLa where the trans-chromosomal 3C signal is
elevated.
The schematic drawings are not necessarily to scale. Like numbers used in the figures
refer to like components, steps and the like. However, it will be understood that the use of a
number to refer to a component in a given figure is not intended to limit the component in
another figure labeled with the same number. In addition, the use of different numbers to refer to
components is not intended to indicate that the different numbered components cannot be the
same or similar to other numbered components.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
As used herein, the terms "organism," "subject," are used interchangeably and refer to
animals and plants. An example of an animal is a mammal, such as a human.
As used herein, the term "cell type" is intended to identify cells based on morphology,
phenotype, developmental origin or other known or recognizable distinguishing cellular
characteristic. A variety of different cell types can be obtained from a single organism (or from
the same species of organism). Exemplary cell types include, but are not limited to, urinary
bladder, pancreatic epithelial, pancreatic alpha, pancreatic beta, pancreatic endothelial, bone
marrow lymphoblast, bone marrow B lymphoblast, bone marrow macrophage, bone marrow
erythroblast, bone marrow dendritic, bone marrow adipocyte, bone marrow osteocyte, bone
marrow chondrocyte, promyeloblast, bone marrow megakaryoblast, bladder, brain B
lymphocyte, brain glial, neuron, brain astrocyte, neuroectoderm, brain macrophage, brain
microglia, brain epithelial, cortical neuron, brain fibroblast, breast epithelial, colon epithelial,
colon B lymphocyte, mammary epithelial, mammary myoepithelial, mammary fibroblast, colon
enterocyte, cervix epithelial, ovary epithelial, ovary fibroblast, breast duct epithelial, tongue
epithelial, tonsil dendritic, tonsil B lymphocyte, peripheral blood lymphoblast, peripheral blood
T lymphoblast, peripheral blood cutaneous T lymphocyte, peripheral blood natural killer,
peripheral blood B lymphoblast, peripheral blood monocyte, peripheral blood myeloblast,
peripheral blood monoblast, peripheral blood promyeloblast, peripheral blood macrophage,
peripheral blood basophil, liver endothelial, liver mast, liver epithelial, liver B lymphocyte,
spleen endothelial, spleen epithelial, spleen B lymphocyte, liver hepatocyte, liver fibroblast, lung
epithelial, bronchus epithelial, lung fibroblast, lung B lymphocyte, lung Schwann, lung
squamous, lung macrophage, lung osteoblast, neuroendocrine, lung alveolar, stomach epithelial,
and stomach fibroblast.
As used herein, the term "tissue" is intended to mean a collection or aggregation of cells
that act together to perform one or more specific functions in an organism. The cells can
optionally be morphologically similar. Exemplary tissues include, but are not limited to, eye,
muscle, skin, tendon, vein, artery, blood, heart, spleen, lymph node, bone, bone marrow, lung,
bronchi, trachea, gut, small intestine, large intestine, colon, rectum, salivary gland, tongue, gall
bladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonad,
testicle, ovary, uterus, fallopian tube, thymus, pituitary, thyroid, adrenal, or parathyroid. Tissue
can be derived from any of a variety of organs of a human or other organism. A tissue can be a
healthy tissue or an unhealthy tissue. Examples of unhealthy tissues include, but are not limited
to, malignancies in lung, breast, colorectum, prostate, nasopharynx, stomach, testes, skin,
nervous system, bone, ovary, liver, hematologic tissues, pancreas, uterus, kidney, lymphoid
tissues, etc. The malignancies may be of a variety of histological subtypes, for example,
carcinoma, adenocarcinoma, sarcoma, fibroadenocarcinoma, neuroendocrine, or undifferentiated.
As used herein, the term "nucleosome" refers to the basic repeating unit of chromatin.
The human genome consists of several meters of DNA compacted within the nucleus of a cell
having an average diameter of -10 p.m. In the eukaryote nucleus, DNA is packaged into a
nucleoprotein complex known as chromatin. The nucleosome (the basic repeating unit of
chromatin) typically includes -146 base pairs of DNA wrapped approximately 1.7 times around
a core histone octamer. The histone octamer consists of two copies of each of the histones H2A,
H2B, H3 and H4. Nucleosomes are regularly spaced along the DNA in the manner of beads on a
string.
As used herein, the term "compartment" is intended to mean an area or volume that
separates or isolates something from other things. Exemplary compartments include, but are not
limited to, vials, tubes, wells, droplets, boluses, beads, vessels, surface features, or areas or
volumes separated by physical forces such as fluid flow, magnetism, electrical current or the
like. In one embodiment, a compartment is a well of a multi-well plate, such as a 96- or 384 -
well plate.
As used herein, a "transposome complex" refers to an integration enzyme and a nucleic
acid including an integration recognition site. A "transposome complex" is a functional complex
formed by a transposase and a transposase recognition site that is capable of catalyzing a
transposition reaction (see, for instance, Gunderson et al., ). Examples of
integration enzymes include, but are not limited to, an integrase or a transposase. Examples of
integration recognition sites include, but are not limited to, a transposase recognition site.
As used herein, the term "nucleic acid" is intended to be consistent with its use in the art
and includes naturally occurring nucleic acids or functional analogs thereof. Particularly useful
functional analogs are capable of hybridizing to a nucleic acid in a sequence specific fashion or
capable of being used as a template for replication of a particular nucleotide sequence. Naturally
occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog
structure can have an alternate backbone linkage including any of a variety of those known in the
art. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g. found in
deoxyribonucleic acid (DNA)) or a ribose sugar (e.g. found in ribonucleic acid (RNA)). A
nucleic acid can contain any of a variety of analogs of these sugar moieties that are known in the
art. A nucleic acid can include native or non-native bases. In this regard, a native
deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine,
thymine, cytosine or guanine and a ribonucleic acid can have one or more bases selected from
the group consisting of adenine, uracil, cytosine or guanine. Useful non-native bases that can be
included in a nucleic acid are known in the art. Examples of non-native bases include a locked
nucleic acid (LNA) and a bridged nucleic acid (BNA). LNA and BNA bases can be incorporated
into a DNA oligonucleotide and increase oligonucleotide hybridization strength and specificity.
LNA and BNA bases and the uses of such bases are known to the person skilled in the art and are
routine.
As used herein, "nuclease" refers to any enzyme that cleaves nucleic acids. Nucleases
belong to a class of enzymes called hydrolases and are usually specific in action, ribonucleases
acting preferentially upon ribonucleic acids (RNA) and deoxyribonucleases acting preferentially
upon deoxyribonucleic acids (DNA).
As used herein, the term "target," when used in reference to a nucleic acid, is intended as
a semantic identifier for the nucleic acid in the context of a method or composition set forth
herein and does not necessarily limit the structure or function of the nucleic acid beyond what is
otherwise explicitly indicated. A target nucleic acid may be essentially any nucleic acid of
known or unknown sequence. It may be, for example, a fragment of genomic DNA or cDNA.
Sequencing may result in determination of the sequence of the whole, or a part of the target
molecule. The targets can be derived from a primary nucleic acid sample, such as a nucleus. The
targets can also be obtained from a primary RNA sample by reverse transcription into cDNA. In
one embodiment, the targets can be processed into templates suitable for amplification by the
placement of universal sequences at the ends of each target fragment.
As used herein, the term "universal," when used to describe a nucleotide sequence, refers
to a region of sequence that is common to two or more nucleic acid molecules where the
molecules also have regions of sequence that differ from each other. A universal sequence that is
present in different members of a collection of molecules can allow capture of multiple different
nucleic acids using a population of universal capture nucleic acids, e.g., capture oligonucleotides
that are complementary to a portion of the universal sequence, e.g., a universal capture sequence.
Non-limiting examples of universal capture sequences include sequences that are identical to or
complementary to P5 and P7 primers. Similarly, a universal sequence present in different
members of a collection of molecules can allow the amplification or replication (e.g.,
sequencing) of multiple different nucleic acids using a population of universal primers that are
complementary to a portion of the universal sequence, e.g., a universal anchor sequence. A
capture oligonucleotide or a universal primer therefore includes a sequence that can hybridize
specifically to a universal sequence. Two universal sequences that hybridize are referred to as a
universal binding pair. For instance, a capture oligonucleotide and a universal capture sequence
that hybridize are a universal binding pair.
The terms "P5" and "P7" may be used when referring to a universal capture sequence or
a capture oligonucleotide. The terms "P5' " (P5 prime) and "P7' " (P7 prime) refer to the
complement of P5 and P7, respectively. It will be understood that any suitable universal capture
sequence or a capture oligonucleotide can be used in the methods presented herein, and that the
use of P5 and P7 are exemplary embodiments only. Uses of capture oligonucleotides such as P5
and P7 or their complements on flowcells are known in the art, as exemplified by the disclosures
of , , , , WO
1998/044151, and . For example, any suitable forward amplification primer,
whether immobilized or in solution, can be useful in the methods presented herein for
hybridization to a complementary sequence and amplification of a sequence. Similarly, any
suitable reverse amplification primer, whether immobilized or in solution, can be useful in the
methods presented herein for hybridization to a complementary sequence and amplification of a
sequence. One of skill in the art will understand how to design and use primer sequences that are
suitable for capture and/or amplification of nucleic acids as presented herein.
As used herein, the term "primer" and its derivatives refer generally to any nucleic acid
that can hybridize to a target sequence of interest. Typically, the primer functions as a substrate
onto which nucleotides can be polymerized by a polymerase; in some embodiments, however,
the primer can become incorporated into the synthesized nucleic acid strand and provide a site to
which another primer can hybridize to prime synthesis of a new strand that is complementary to
the synthesized nucleic acid molecule. The primer can include any combination of nucleotides or
analogs thereof. In some embodiments, the primer is a single-stranded oligonucleotide or
polynucleotide. The terms "polynucleotide" and "oligonucleotide" are used interchangeably
herein to refer to a polymeric form of nucleotides of any length, and may include
ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The terms should be
understood to include, as equivalents, analogs of either DNA or RNA made from nucleotide
analogs and to be applicable to single stranded (such as sense or anti sense) and double stranded
polynucleotides. The term as used herein also encompasses cDNA, that is complementary or
copy DNA produced from an RNA template, for example by the action of reverse transcriptase.
This term refers only to the primary structure of the molecule. Thus, the term includes triple-,
double- and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double- and
single-stranded ribonucleic acid ("RNA").
As used herein, the term "adapter" and its derivatives, e.g., universal adapter, refers
generally to any linear oligonucleotide which can be ligated to a nucleic acid molecule of the
disclosure. In some embodiments, the adapter is substantially non-complementary to the 3' end
or the 5' end of any target sequence present in the sample. In some embodiments, suitable
adapter lengths are in the range of about 10-100 nucleotides, about 12-60 nucleotides, or about
15-50 nucleotides in length. Generally, the adapter can include any combination of nucleotides
and/or nucleic acids. In some aspects, the adapter can include one or more cleavable groups at
one or more locations. In another aspect, the adapter can include a sequence that is substantially
identical, or substantially complementary, to at least a portion of a primer, for example a
universal primer. In some embodiments, the adapter can include a barcode (also referred to
herein as a tag or index) to assist with downstream error correction, identification, or sequencing.
The terms "adaptor" and "adapter" are used interchangeably.
As used herein, the term "each," when used in reference to a collection of items, is
intended to identify an individual item in the collection but does not necessarily refer to every
item in the collection unless the context clearly dictates otherwise.
As used herein, the term "transport" refers to movement of a molecule through a fluid.
The term can include passive transport such as movement of molecules along their concentration
gradient (e.g. passive diffusion). The term can also include active transport whereby molecules
can move along their concentration gradient or against their concentration gradient. Thus,
transport can include applying energy to move one or more molecule in a desired direction or to
a desired location such as an amplification site.
As used herein, "amplify", "amplifying" or "amplification reaction" and their derivatives,
refer generally to any action or process whereby at least a portion of a nucleic acid molecule is
replicated or copied into at least one additional nucleic acid molecule. The additional nucleic
acid molecule optionally includes sequence that is substantially identical or substantially
complementary to at least some portion of the template nucleic acid molecule. The template
nucleic acid molecule can be single-stranded or double-stranded and the additional nucleic acid
molecule can independently be single-stranded or double-stranded. Amplification optionally
includes linear or exponential replication of a nucleic acid molecule. In some embodiments, such
amplification can be performed using isothermal conditions; in other embodiments, such
amplification can include thermocycling. In some embodiments, the amplification is a multiplex
amplification that includes the simultaneous amplification of a plurality of target sequences in a
single amplification reaction. In some embodiments, "amplification" includes amplification of at
least some portion of DNA and RNA based nucleic acids alone, or in combination. The
amplification reaction can include any of the amplification processes known to one of ordinary
skill in the art. In some embodiments, the amplification reaction includes polymerase chain
reaction (PCR).
As used herein, "amplification conditions" and its derivatives, generally refers to
conditions suitable for amplifying one or more nucleic acid sequences. Such amplification can be
linear or exponential. In some embodiments, the amplification conditions can include isothermal
conditions or alternatively can include thermocycling conditions, or a combination of isothermal
and thermocycling conditions. In some embodiments, the conditions suitable for amplifying one
or more nucleic acid sequences include polymerase chain reaction (PCR) conditions. Typically,
the amplification conditions refer to a reaction mixture that is sufficient to amplify nucleic acids
such as one or more target sequences flanked by a universal sequence, or to amplify an amplified
target sequence ligated to one or more adapters. Generally, the amplification conditions include a
catalyst for amplification or for nucleic acid synthesis, for example a polymerase; a primer that
possesses some degree of complementarity to the nucleic acid to be amplified; and nucleotides,
such as deoxyribonucleotide triphosphates (dNTPs) to promote extension of the primer once
hybridized to the nucleic acid. The amplification conditions can require hybridization or
annealing of a primer to a nucleic acid, extension of the primer and a denaturing step in which
the extended primer is separated from the nucleic acid sequence undergoing amplification.
Typically, but not necessarily, amplification conditions can include thermocycling; in some
embodiments, amplification conditions include a plurality of cycles where the steps of annealing,
extending and separating are repeated. Typically, the amplification conditions include cations
such as Mg' or Mn' and can also include various modifiers of ionic strength.
As used herein, "re-amplification" and their derivatives refer generally to any process
whereby at least a portion of an amplified nucleic acid molecule is further amplified via any
suitable amplification process (referred to in some embodiments as a "secondary" amplification),
thereby producing a reamplified nucleic acid molecule. The secondary amplification need not be
identical to the original amplification process whereby the amplified nucleic acid molecule was
produced; nor need the reamplified nucleic acid molecule be completely identical or completely
complementary to the amplified nucleic acid molecule; all that is required is that the reamplified
nucleic acid molecule include at least a portion of the amplified nucleic acid molecule or its
complement. For example, the re-amplification can involve the use of different amplification
conditions and/or different primers, including different target-specific primers than the primary
amplification.
As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of
Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, which describe a method for increasing the
concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without
cloning or purification. This process for amplifying the polynucleotide of interest consists of
introducing a large excess of two oligonucleotide primers to the DNA mixture containing the
desired polynucleotide of interest, followed by a series of thermal cycling in the presence of a
DNA polymerase. The two primers are complementary to their respective strands of the double
stranded polynucleotide of interest. The mixture is denatured at a higher temperature first and the
primers are then annealed to complementary sequences within the polynucleotide of interest
molecule. Following annealing, the primers are extended with a polymerase to form a new pair
of complementary strands. The steps of denaturation, primer annealing and polymerase extension
can be repeated many times (referred to as thermocycling) to obtain a high concentration of an
amplified segment of the desired polynucleotide of interest. The length of the amplified segment
of the desired polynucleotide of interest (amplicon) is determined by the relative positions of the
primers with respect to each other, and therefore, this length is a controllable parameter. By
virtue of repeating the process, the method is referred to as the "polymerase chain reaction"
(hereinafter "PCR"). Because the desired amplified segments of the polynucleotide of interest
become the predominant nucleic acid sequences (in terms of concentration) in the mixture, they
are said to be "PCR amplified". In a modification to the method discussed above, the target
nucleic acid molecules can be PCR amplified using a plurality of different primer pairs, in some
cases, one or more primer pairs per target nucleic acid molecule of interest, thereby forming a
multiplex PCR reaction.
As defined herein "multiplex amplification" refers to selective and non-random
amplification of two or more target sequences within a sample using at least one target-specific
primer. In some embodiments, multiplex amplification is performed such that some or all of the
target sequences are amplified within a single reaction vessel. The "plexy" or "plex" of a given
multiplex amplification refers generally to the number of different target-specific sequences that
are amplified during that single multiplex amplification. In some embodiments, the plexy can be
about 12-plex, 24-plex, 48-plex, 96-plex, 192-plex, 384-plex, 768-plex, 1536-plex, 3072-plex,
6144-plex or higher. It is also possible to detect the amplified target sequences by several
different methodologies (e.g., gel electrophoresis followed by densitometry, quantitation with a
bioanalyzer or quantitative PCR, hybridization with a labeled probe; incorporation of
biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-
labeled deoxynucleotide triphosphates into the amplified target sequence).
As used herein, "amplified target sequences" and its derivatives, refers generally to a
nucleic acid sequence produced by the amplifying the target sequences using target-specific
primers and the methods provided herein. The amplified target sequences may be either of the
same sense (i.e. the positive strand) or antisense (i.e., the negative strand) with respect to the
target sequences.
As used herein, the terms "ligating", "ligation" and their derivatives refer generally to the
process for covalently linking two or more molecules together, for example covalently linking
two or more nucleic acid molecules to each other. In some embodiments, ligation includes
joining nicks between adjacent nucleotides of nucleic acids. In some embodiments, ligation
includes forming a covalent bond between an end of a first and an end of a second nucleic acid
molecule. In some embodiments, the ligation can include forming a covalent bond between a 5'
phosphate group of one nucleic acid and a 3' hydroxyl group of a second nucleic acid thereby
forming a ligated nucleic acid molecule. Generally for the purposes of this disclosure, an
amplified target sequence can be ligated to an adapter to generate an adapter-ligated amplified
target sequence.
As used herein, "ligase" and its derivatives, refers generally to any agent capable of
catalyzing the ligation of two substrate molecules. In some embodiments, the ligase includes an
enzyme capable of catalyzing the joining of nicks between adjacent nucleotides of a nucleic acid.
In some embodiments, the ligase includes an enzyme capable of catalyzing the formation of a
covalent bond between a 5' phosphate of one nucleic acid molecule to a 3' hydroxyl of another
nucleic acid molecule thereby forming a ligated nucleic acid molecule. Suitable ligases may
include, but are not limited to, T4 DNA ligase, T4 RNA ligase, and E. coli DNA ligase.
As used herein, "ligation conditions" and its derivatives, generally refers to conditions
suitable for ligating two molecules to each other. In some embodiments, the ligation conditions
are suitable for sealing nicks or gaps between nucleic acids. As used herein, the term nick or gap
is consistent with the use of the term in the art. Typically, a nick or gap can be ligated in the
presence of an enzyme, such as ligase at an appropriate temperature and pH. In some
embodiments, T4 DNA ligase can join a nick between nucleic acids at a temperature of about 70-
72° C.
The term "flowcell" as used herein refers to a chamber comprising a solid surface across
which one or more fluid reagents can be flowed. Examples of flowcells and related fluidic
systems and detection platforms that can be readily used in the methods of the present disclosure
are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; US
7,057,026; WO 91/06678; WO 07/123744; US 7,329,492; US 7,211,414; US 7,315,019; US
7,405,281, and US 2008/0108082.
As used herein, the term "amplicon," when used in reference to a nucleic acid, means the
product of copying the nucleic acid, wherein the product has a nucleotide sequence that is the
same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. An
amplicon can be produced by any of a variety of amplification methods that use the nucleic acid,
or an amplicon thereof, as a template including, for example, polymerase extension, polymerase
chain reaction (PCR), rolling circle amplification (RCA), ligation extension, or ligation chain
reaction. An amplicon can be a nucleic acid molecule having a single copy of a particular
nucleotide sequence (e.g. a PCR product) or multiple copies of the nucleotide sequence (e.g. a
concatameric product of RCA). A first amplicon of a target nucleic acid is typically a
complementary copy. Subsequent amplicons are copies that are created, after generation of the
first amplicon, from the target nucleic acid or from the first amplicon. A subsequent amplicon
can have a sequence that is substantially complementary to the target nucleic acid or
substantially identical to the target nucleic acid.
As used herein, the term "amplification site" refers to a site in or on an array where one
or more amplicons can be generated. An amplification site can be further configured to contain,
hold or attach at least one amplicon that is generated at the site.
As used herein, the term "array" refers to a population of sites that can be differentiated
from each other according to relative location. Different molecules that are at different sites of an
array can be differentiated from each other according to the locations of the sites in the array. An
individual site of an array can include one or more molecules of a particular type. For example, a
site can include a single target nucleic acid molecule having a particular sequence or a site can
include several nucleic acid molecules having the same sequence (and/or complementary
sequence, thereof). The sites of an array can be different features located on the same substrate.
Exemplary features include without limitation, wells in a substrate, beads (or other particles) in
or on a substrate, projections from a substrate, ridges on a substrate or channels in a substrate.
The sites of an array can be separate substrates each bearing a different molecule. Different
molecules attached to separate substrates can be identified according to the locations of the
substrates on a surface to which the substrates are associated or according to the locations of the
substrates in a liquid or gel. Exemplary arrays in which separate substrates are located on a
surface include, without limitation, those having beads in wells.
As used herein, the term "capacity," when used in reference to a site and nucleic acid
material, means the maximum amount of nucleic acid material that can occupy the site. For
example, the term can refer to the total number of nucleic acid molecules that can occupy the site
in a particular condition. Other measures can be used as well including, for example, the total
mass of nucleic acid material or the total number of copies of a particular nucleotide sequence
that can occupy the site in a particular condition. Typically, the capacity of a site for a target
nucleic acid will be substantially equivalent to the capacity of the site for amplicons of the target
nucleic acid.
As used herein, the term "capture agent" refers to a material, chemical, molecule or
moiety thereof that is capable of attaching, retaining or binding to a target molecule (e.g. a target
nucleic acid). Exemplary capture agents include, without limitation, a capture nucleic acid (also
referred to herein as a capture oligonucleotide) that is complementary to at least a portion of a
target nucleic acid, a member of a receptor-ligand binding pair (e.g. avidin, streptavidin, biotin,
lectin, carbohydrate, nucleic acid binding protein, epitope, antibody, etc.) capable of binding to a
target nucleic acid (or linking moiety attached thereto), or a chemical reagent capable of forming
a covalent bond with a target nucleic acid (or linking moiety attached thereto).
As used herein, the term "clonal population" refers to a population of nucleic acids that is
homogeneous with respect to a particular nucleotide sequence. The homogenous sequence is
typically at least 10 nucleotides long, but can be even longer including for example, at least 50,
100, 250, 500 or 1000 nucleotides long. A clonal population can be derived from a single target
nucleic acid or template nucleic acid. Typically, all of the nucleic acids in a clonal population
will have the same nucleotide sequence. It will be understood that a small number of mutations
(e.g. due to amplification artifacts) can occur in a clonal population without departing from
clonality.
As used herein, "providing" in the context of a composition, an article, a nucleic acid, or
a nucleus means making the composition, article, nucleic acid, or nucleus, purchasing the
composition, article, nucleic acid, or nucleus, or otherwise obtaining the compound,
composition, article, or nucleus.
The term "and/or" means one or all of the listed elements or a combination of any two or
more of the listed elements.
The words "preferred" and "preferably" refer to embodiments of the disclosure that may
afford certain benefits, under certain circumstances. However, other embodiments may also be
preferred, under the same or other circumstances. Furthermore, the recitation of one or more
preferred embodiments does not imply that other embodiments are not useful, and is not intended
to exclude other embodiments from the scope of the disclosure.
The terms "comprises" and variations thereof do not have a limiting meaning where these
terms appear in the description and claims.
It is understood that wherever embodiments are described herein with the language
"include," "includes," or "including," and the like, otherwise analogous embodiments described
in terms of "consisting of and/or "consisting essentially of are also provided.
Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably
and mean one or more than one.
Also herein, the recitations of numerical ranges by endpoints include all numbers
subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
For any method disclosed herein that includes discrete steps, the steps may be conducted
in any feasible order. And, as appropriate, any combination of two or more steps may be
conducted simultaneously.
Reference throughout this specification to "one embodiment," "an embodiment," "certain
embodiments," or "some embodiments," etc., means that a particular feature, configuration,
composition, or characteristic described in connection with the embodiment is included in at
least one embodiment of the disclosure. Thus, the appearances of such phrases in various places
throughout this specification are not necessarily referring to the same embodiment of the
disclosure. Furthermore, the particular features, configurations, compositions, or characteristics
may be combined in any suitable manner in one or more embodiments.
The method provided herein can be used to produce sequencing libraries that include the
whole genomes of a plurality of single cells. In one embodiment, the method can be used to
detect copy number variants (CNV, e.g., the number of copies of a particular sequence, such as a
gene, in the genotype of a cell). For instance, the method can be used to quantify the frequency
of CNV-harboring nuclei in a sample of somatic cells from an organism, or provide information
on heterogeneity in the context of certain conditions, such as cancer.
The method provided herein includes providing isolated nuclei from a plurality of cells
( block 12; block 12). The cells can be from any organism(s), and from any cell
type or any tissue of the organism(s). The method can further include dissociating cells, and/or
isolating the nuclei. Methods for isolating nuclei from cells are known to the person skilled in
the art and are routine. The number of nuclei can be at least two. The upper limit is dependent
on the practical limitations of equipment (e.g., multi-well plates) used in other steps of the
method as described herein. For instance, in one embodiment the number of nuclei can be no
greater than 1,000,000,000, no greater than 100,000,000, no greater than 10,000,000, no greater
than 1,000,000, no greater than 100,000, no greater than 10,000, or no greater than 1,000. The
skilled person will recognize that the nucleic acid molecules in each nucleus represent the entire
genetic complement of an organism (also referred to as the whole genome of an organism), and
are genomic DNA molecules which include both intron and exon sequences, as well as non-
coding regulatory sequences such as promoter and enhancer sequences.
The isolated nuclei can be nucleosome-free, or can be subjected to conditions that deplete
the nuclei of nucleosomes, generating nucleosome-depleted nuclei ( block 13;
block 13). Nucleosome-depleted nuclei are useful in methods for determining the DNA
sequence of the whole genome of a cell.
In one embodiment, the conditions used for nucleosome-depletion maintain the integrity
of the isolated nuclei. Typically, nucleosome-depletion methods are used on a pellet or
suspension of single cells, thus in those embodiments where an adherent cell culture or tissue is
used as a source of the cells, the source is treated to obtain a pellet or suspension of single cells.
In one embodiment, the conditions for nucleosome-depletion include a chemical
treatment with a chaotropic agent capable of disrupting nucleic acid-protein interactions. An
example of a useful chaotropic agent includes, but is not limited to, 3,5-lithium diiodosalicylic
acid. Conditions for using 3,5-lithium diiodosalicylic acid include adding it to a pellet of cells
and incubating on ice.
In another embodiment, the conditions include a chemical treatment with a detergent
capable of disrupting nucleic acid-protein interactions. An example of a useful detergent
includes, but is not limited to, sodium dodecyl sulfate (SDS). Conditions for using SDS include
adding it to a pellet of cells and incubating at an elevated temperature such as 42°C, and then
adding a nonionic detergent such as TritonTm X-100 and incubating at an elevated temperature
such as 42°C.
In some embodiments, when a detergent such as SDS is used, the nuclei are exposed to a
cross-linking agent prior to the depletion of nucleosomes. In one embodiment, the nuclei are
exposed to the cross-linking agent while inside cells (, block 11), and in another
embodiment, isolated nuclei are exposed to the cross-linking agent. A useful example of a cross-
linking agent includes, but is not limited to, formaldehyde (Hoffman et al., 2015, J. Biol. Chem.,
290:26404-26411). Treatment of cells with formaldehyde can include adding formaldehyde to a
suspension of cells and incubating at room temperature. In one embodiment, the concentration
of formaldehyde can be from 0.2% to 2%, such as greater than 0.2% and no greater than 1.5%.
After the formaldehyde treatment, the nuclei can be exposed to glycine and a nonionic, non-
denaturing detergent nonionic, non-denaturing detergent such as Igepal®. If cells are cross-
linked before isolating the nuclei, the cross-linking, can be, and typically is, reversed by
incubation at 55°C to 72°C, such as 68°C, for 30 minutes to 16 hours, such as 1 hour (,
block 19). Reversal typically occurs later, after distributing subsets of pooled indexed nuclei into
a second plurality of compartments (, block 18) and before generating dual-index
fragments (, block 20). The distributing subsets and generating dual-index fragments is
described herein.
In some embodiments where a cross-linking agent is used, the method can also include
manipulations that provide information on chromosome structure within a nucleus, such as
chromatin folding analysis and detection of genomic rearrangements such as, but not limited to,
translocations. Such types of analyses are known in art as chromosome conformation capture
(3C) and related methods (4C, 5C, and Hi-C). The manipulations typically include digestion of
genomic DNA within a nucleus (, block 14) followed by ligation of the ends of genomic
fragments that are in close proximity (, block 15). These steps result in chimeric
fragments, where the chimeric fragments are likely nearby in physical proximity within the
nucleus which are also typically near in sequence space (Nagano et al., 2013, Nature, 502:59-
64). Typically, after nuclei are exposed to a cross-linking agent and before fragmenting nucleic
acids, the genomic DNA present in the nuclei is digested with a nuclease, such as a restriction
endonuclease (, block 14). Any restriction endonuclease can be used, and in one
embodiment, the restriction endonuclease cleaves a nucleic acid to result in two overhangs, also
known to the skilled person as sticky ends. After digestion of the genomic DNA with a
restriction endonuclease, the nuclei are exposed to a ligase to join fragments of genomic DNA
(, block 15).
During the process of depleting nucleosomes in the isolated nuclei ( block 13;
block 13), the integrity of the isolated nuclei is maintained. Whether nuclei remain
intact after exposure to conditions for depleting nucleosomes can be determined by visualizing
the status of the nuclei by routine methods such as phase-contrast imaging. In one embodiment,
at least 100,000 nuclei are intact after nucleosome-depletion.
The method provided herein includes distributing subsets of the nucleosome-depleted
nuclei into a first plurality of compartments ( block 14; , block 16). The
number of nuclei present in a subset, and therefore in each compartment, can be at least 1. In
one embodiment, the number of nuclei present in a subset is no greater than 1,000,000, no
greater than 100,000, no greater than 10,000, no greater than 4,000, no greater than 3,000, no
greater than 2,000, or no greater than 1,000. In one embodiment, the number of nuclei present in
a subset can be 1 to 1,000, 1,000 to 10,000, 10,000 to 100,000, or 100,000 to 1,000,000. In one
embodiment, the number of nuclei present each subset is approximately equal. Methods for
distributing nuclei into subsets are known to the person skilled in the art and are routine.
Examples include, but are not limited to, fluorescence-activated nuclei sorting (FANS).
Each compartment includes a transposome complex. The transposome complex can be
added to each compartment before, after, or at the same time a subset of the nuclei is added to
the compartment. The transposome complex, a transposase bound to a transposase recognition
site, can insert the transposase recognition site into a target nucleic acid within a nucleus in a
process sometimes termed "tagmentation." In some such insertion events, one strand of the
transposase recognition site may be transferred into the target nucleic acid. Such a strand is
referred to as a "transferred strand." In one embodiment, a transposome complex includes a
dimeric transposase having two subunits, and two non-contiguous transposon sequences. In
another embodiment, a transposase includes a dimeric transposase having two subunits, and a
contiguous transposon sequence.
Some embodiments can include the use of a hyperactive Tn5 transposase and a Tn5-type
transposase recognition site (Goryshin and Reznikoff, I Biol. Chem., 273:7367 (1998)), or MuA
transposase and a Mu transposase recognition site comprising R1 and R2 end sequences
(Mizuuchi, K., Cell, 35: 785, 1983; Savilahti, H, et al., EMBO J., 14: 4893, 1995). Tn5 Mosaic
End (ME) sequences can also be used as optimized by a skilled artisan.
More examples of transposition systems that can be used with certain embodiments of the
compositions and methods provided herein include Staphylococcus aureus Tn552 (Colegio et al.,
1 Bacteriol., 183: 2384-8, 2001; Kirby C et al., Mol. Microbiol., 43: 173-86, 2002), Tyl (Devine
& Boeke, Nucleic Acids Res., 22: 3765-72, 1994 and International Publication WO 95/23875),
Transposon Tn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, Review in: Curr Top
Microbiol Immunol., 204:27-48, 1996), Tn/O and IS10 (Kleckner N, et al., Curr Top Microbiol
Immunol., 204:49-82, 1996), Mariner transposase (Lampe D J, et al., EMBO 1, 15: 5470-9,
1996), Tcl (Plasterk R H, Curr. Topics Microbiol. Immunol., 204: 125-43, 1996), P Element
(Gloor, G B, Methods Mol. Biol., 260: 97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem.
265:18829-32, 1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr. Top. Microbiol.
Immunol. 204: 1-26, 1996), retroviruses (Brown, et al., Proc Natl Acad Sci USA, 86:2525-9,
1989), and retrotransposon of yeast (Boeke & Corces, Annu Rev Microbiol. 43:403-34, 1989).
More examples include IS5, Tnl 0, Tn903, IS911, and engineered versions of transposase family
enzymes (Zhang et al., (2009) PLoS Genet. 5:e1000689. Epub 2009 Oct 16; Wilson C. et al
(2007) J Microbiol. Methods 71:332-5).
Other examples of integrases that may be used with the methods and compositions
provided herein include retroviral integrases and integrase recognition sequences for such
retroviral integrases, such as integrases from HIV-1, HIV-2, SIV, PFV-1, RSV.
Transposon sequences useful with the methods and compositions described herein are
provided in U.S. Patent Application Pub. No. 2012/0208705, U.S. Patent Application Pub. No.
2012/0208724 and Int. Patent Application Pub. No. . In some embodiments, a
transposon sequence includes a first transposase recognition site, a second transposase
recognition site, and an index sequence present between the two transposase recognition sites.
Some transposome complexes useful herein include a transposase having two transposon
sequences. In some such embodiments, the two transposon sequences are not linked to one
another, in other words, the transposon sequences are non-contiguous with one another.
Examples of such transposomes are known in the art (see, for instance, U.S. Patent Application
Pub. No. 2010/0120098).
In some embodiments, a transposome complex includes a transposon sequence nucleic
acid that binds two transposase subunits to form a "looped complex" or a "looped transposome."
In one example, a transposome includes a dimeric transposase and a transposon sequence.
Looped complexes can ensure that transposons are inserted into target DNA while maintaining
ordering information of the original target DNA and without fragmenting the target DNA. As
will be appreciated, looped structures may insert desired nucleic acid sequences, such as indexes,
into a target nucleic acid, while maintaining physical connectivity of the target nucleic acid. In
some embodiments, the transposon sequence of a looped transposome complex can include a
fragmentation site such that the transposon sequence can be fragmented to create a transposome
complex comprising two transposon sequences. Such transposome complexes are useful to
ensuring that neighboring target DNA fragments, in which the transposons insert, receive code
combinations that can be unambiguously assembled at a later stage of the assay.
A transposome complex also includes at least one index sequence, also referred to as a
transposase index. The index sequence is present as part of the transposon sequence. In one
embodiment, the index sequence can be present on a transferred strand, the strand of the
transposase recognition site that is transferred into the target nucleic acid. An index sequence,
also referred to as a tag or barcode, is useful as a marker characteristic of the compartment in
which a particular target nucleic acid was present. The index sequence of a transposome
complex is different for each compartment. Accordingly, in this embodiment, an index is a
nucleic acid sequence tag which is attached to each of the target nucleic acids present in a
particular compartment, the presence of which is indicative of, or is used to identify, the
compartment in which a population of nuclei were present at this stage of the method.
An index sequence can be up to 20 nucleotides in length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20. A four nucleotide tag gives a possibility of multiplexing
256 samples on the same array, and a six base tag enables 4096 samples to be processed on the
same array.
In one embodiment, the transferred strand can also include a universal sequence.
Universal sequences are described herein. Thus, in some embodiments where the transferred
strand is transferred to target nucleic acids, the target nucleic acids include a transposase index a
universal sequence, or a combination thereof.
The method also includes generating indexed nuclei ( block 15; block 17).
In one embodiment, generating indexed nuclei includes fragmenting nucleic acids present in the
subsets of nucleosome-depleted nuclei (e.g., the nuclei acids present in each compartment) into a
plurality of nucleic acid fragments. After nucleic acids are fragmented, the transposase remains
attached to the nucleic acid fragments, such that nucleic acid fragments derived from the same
genomic DNA molecule remain physically linked (Adey et al., 2014, Genome Res., 24:2041-
2049).
In one embodiment, fragmenting nucleic acids is accomplished by using a fragmentation
site present in the nucleic acids. Typically, fragmentation sites are introduced into target nucleic
acids by using a transposome complex. For instance, a looped transposome complex can include
a fragmentation site. A fragmentation site can be used to cleave the physical, but not the
informational association between index sequences that have been inserted into a target nucleic
acid. Cleavage may be by biochemical, chemical or other means. In some embodiments, a
fragmentation site can include a nucleotide or nucleotide sequence that may be fragmented by
various means. Examples of fragmentation sites include, but are not limited to, a restriction
endonuclease site, at least one ribonucleotide cleavable with an RNAse, nucleotide analogues
cleavable in the presence of a certain chemical agent, a diol linkage cleavable by treatment with
periodate, a disulfide group cleavable with a chemical reducing agent, a cleavable moiety that
may be subject to photochemical cleavage, and a peptide cleavable by a peptidase enzyme or
other suitable means (see, for instance, U.S. Patent Application Pub. No. 2012/0208705, U.S.
Patent Application Pub. No. 2012/0208724 and . The result of the fragmenting
is a population of indexed nuclei, where each nucleus contains indexed nucleic acid fragments.
The indexed nucleic acid fragments can, and typically do, include on at least one strand the index
sequence indicative of the particular compartment. An example of an indexed nucleic acid
fragment is shown in The single strand of the indexed nucleic acid fragment 20 includes
nucleotides 21 and 22 originating from the transferred strand of the transposome complex, which
includes a transposase index and a universal sequence that can be used for amplification and/or
sequencing. The indexed nucleic acid fragment also includes the nucleotides originating from
the genomic DNA of a nucleus 23.
The indexed nuclei from multiple compartments can be combined ( block 16; block 18). For instance, the indexed nuclei from 2 to 96 compartments (when a 96-well plate
is used), or from 2 to 384 compartments (when a 384-well plate is used) are combined. Subsets
of these combined indexed nuclei, referred to herein as pooled indexed nuclei, are then
distributed into a second plurality of compartments. The number of nuclei present in a subset,
and therefor in each compartment, is based in part on the desire to reduce index collisions, which
is the presence of two nuclei having the same transposase index ending up in the same
compartment in this step of the method. The number of nuclei present in a subset in this
embodiment can be from 2 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In one embodiment, the number of nuclei
present in a subset is from 20 to 24, such as 22. In one embodiment, the number of nuclei
present each subset is approximately equal. In one embodiment, the number of nuclei present
each subset is at least 10 times fewer nuclei than the subsets of the nucleosome-depleted nuclei
( block 14; block 16). In one embodiment, the number of nuclei present each
subset is at least 100 times fewer nuclei than the subsets of the nucleosome-depleted nuclei ( 1, block 14; block 16). Methods for distributing nuclei into subsets are known to the
person skilled in the art and are routine. Examples include, but are not limited to, fluorescence-
activated nuclei sorting (FANS).
Distribution of nuclei into subsets is followed by incorporating into the indexed nucleic
acid fragments in each compartment a second index sequence to generate dual-index fragments,
where the second index sequence in each compartment is different from second index sequences
in the other compartments. This results in the further indexing of the indexed nucleic acid
fragments ( block 17; block 20) prior to immobilizing and sequencing. In those
embodiments where cells are cross-linked by a cross-linking agent, the transposases attached to
the indexed nucleic acid fragments are dissociated from the indexed nucleic acid fragments. In
one embodiment, the attached transposases are dissociated before the cross-linking is reversed
(, block 19). A detergent can be used to dissociate the transposases, and in one
embodiment the detergent is sodium dodecyl sulfate (SDS).
In one embodiment, the incorporation is typically by an exponential amplification
reaction, such as a PCR. The universal sequences present at ends of the indexed nucleic acid
fragment can be used for the binding of universal anchor sequences which can serve as primers
and be extended in an amplification reaction. Typically, two different universal primers are
used. One primer hybridizes with universal sequences at the 3' end of one strand of the indexed
nucleic acid fragments, and a second primer hybridizes with universal sequences at the 3' end of
the other strand of the indexed nucleic acid fragments. Thus, the anchor sequence of each primer
can be different. Suitable primers can each include additional universal sequences, such as a
universal capture sequence, and another index sequence. Because each primer can include an
index, this step results in the addition of one or two index sequences, e.g., a second and an
optional third index. Indexed nucleic acid fragments having the second and the optional third
indexes are referred to as dual-index fragments. The second and third indexes can be the reverse
complements of each other, or the second and third indexes can have sequences that are not the
reverse complements of each other. This second index sequence and optional third index is
unique for each compartment in which the distributed indexed nuclei were placed ( block
16; block 18).
In one embodiment, the incorporation of the second index sequence includes contacting
the indexed nucleic acid fragments in each compartment with a first universal primer and a
second universal primer. The first universal primer includes a sequence identical to a portion of
the first universal sequence, and the second universal primer includes a sequence complementary
to a portion of the first universal sequence. Each primer includes an index sequence. In one
embodiment, the index sequence of the first universal primer is the reverse complement of the
index sequence of the second universal primer. In another embodiment, the index sequence of
the first universal primer is different from the reverse complement of the index sequence of the
second universal primer.
In one embodiment, the first universal primer also includes a first capture sequence and a
first anchor sequence complementary to a universal sequence at the 3' end of the dual-index
fragments. In one embodiment, the first capture sequence includes the P5 primer sequence. In
one embodiment, the second universal primer also includes a second capture sequence and a
second anchor sequence complementary to a universal sequence at the 5' end of the dual-index
fragments. In one embodiment, the second capture sequence includes the reverse complement of
the P7 primer sequence.
In another embodiment, the incorporation includes subjecting the indexed nucleic acid
fragments to conditions that result in the ligation of additional sequences to both ends of the
fragments. In one embodiment, blunt-ended ligation can be used. In another embodiment, the
fragments are prepared with single overhanging nucleotides by, for example, activity of certain
types of DNA polymerase such as Taq polymerase or Klenow exo minus polymerase which has
a non-template-dependent terminal transferase activity that adds a single deoxynucleotide, for
example, deoxyadenosine (A) to the 3' ends of the indexed nucleic acid fragments. Such
enzymes can be used to add a single nucleotide 'A' to the blunt ended 3' terminus of each strand
of the fragments. Thus, an 'A' could be added to the 3' terminus of each strand of the double-
stranded target fragments by reaction with Taq or Klenow exo minus polymerase, while the
additional sequences to be added to each end of the fragment can include a compatible 'T'
overhang present on the 3' terminus of each region of double stranded nucleic acid to be added.
This end modification also prevents self-ligation of the nucleic acids such that there is a bias
towards formation of the indexed nucleic acid fragments flanked by the sequences that are added
in this embodiment.
Fragmentation of nucleic acid molecules by the methods described herein can result in
fragments with a heterogeneous mix of blunt and 3'- and 5'-overhanging ends. In some
embodiments, it is therefore desirable to repair the fragment ends using methods or kits (such as
the Lucigen DNA terminator End Repair Kit) known in the art to generate ends that are optimal
for insertion, for example, into blunt sites of cloning vectors. In a particular embodiment, the
fragment ends of the population of nucleic acids are blunt ended. More particularly, the fragment
ends are blunt ended and phosphorylated. The phosphate moiety can be introduced via enzymatic
treatment, for example, using polynucleotide kinase.
In one embodiment, the indexed nucleic acid fragments are treated by first ligating
identical universal adapters (also referred to as 'mismatched adaptors,' the general features of
which are described in Gormley et al., US 7,741,463, and Bignell et al., US 8,053,192,) to the 5'
and 3' ends of the indexed nucleic acid fragments to form dual-index fragments. In one
embodiment, the universal adaptor includes all sequences necessary for sequencing, including
one or two index sequences and sequences for immobilizing the dual-index fragments on an
array. Because the nucleic acids to be sequenced are from single cells, further amplification of
the dual-index fragments is helpful to achieve a sufficient number of dual-index fragments for
sequencing.
In one embodiment, the incorporation of the second index sequence includes ligating a
universal adapter to the indexed nucleic acid fragments in each compartment. The universal
adapter includes two nucleic acid strands, wherein each strand includes the second index
sequence. In one embodiment, the second index sequence of one strand of the universal adapter
is the reverse complement of the second index sequence of the second strand of the universal
adapter. In other embodiment, the second index sequence of one strand of the universal adapter
is different from the reverse complement of the second index sequence of the second strand of
the universal adapter.
In one embodiment, the universal adapter also includes a first capture sequence and a first
anchor sequence. In one embodiment, the first capture sequence includes the P5 primer
sequence. In one embodiment, the universal adapter also includes a second capture sequence and
a second anchor sequence. In one embodiment, the second capture sequence includes the reverse
complement of the P7 primer sequence.
In another embodiment, when the universal adapter ligated to the indexed nucleic acid
fragments does not include all sequences necessary for sequencing, then an exponential
amplification step, such as PCR, can be used to further modify the universal adapters present in
each indexed nucleic acid fragment prior to immobilizing and sequencing. For instance, an
initial primer extension reaction is carried out using a universal anchor sequence complementary
to a universal sequence present in the indexed nucleic acid fragment, in which extension
products complementary to both strands of each individual indexed nucleic acid fragment are
formed. Typically, the PCR adds additional universal sequences, such as a universal capture
sequence, and another index sequence. Because each primer can include an index, this step
results in the addition of one or two index sequences, e.g., a second and an optional third index,
and indexing of the indexed nucleic acid fragment by adapter ligation ( block 17;
block 20).
After the universal adapters are added, either by a single step method of ligating a
universal adaptor including all sequences necessary for sequencing, or by a two-step method of
ligating a universal adapter and then an exponential amplification to further modify the universal
adapter, the final dual-index fragments will include a universal capture sequence, a second index
sequence, and an optional third index sequence. The second and third indexes can be the reverse
complements of each other, or the second and third indexes can have sequences that are not the
reverse complements of each other. These second and optional third index sequences are unique
for each compartment in which the distributed indexed nuclei were placed ( block 17;
block 20) after the first index was added by tagmentation. The result of adding universal
adapters to each end is a plurality or library of dual-index fragments having a structure similar or
identical to the dual-index fragment 30 shown in A single strand of the dual-index
fragment 30 includes a capture sequence 31 and 38, also referred to as a 3' flowcell adapter (e.g.,
P5) and 5' flowcell adapter (e.g., P7'), respectively, and an index 32 and 37, such as i5 and i7.
The dual-index fragment 30 also includes nucleotides originating from the transferred strand of
the transposome complex 33, which includes a transposase index 34 and a universal sequence 35
that can be used for amplification and/or sequencing. The dual-index fragment also includes the
nucleotides originating from the genomic DNA of a nucleus 36.
The resulting dual-index fragments collectively provide a library of nucleic acids that can
be immobilized and then sequenced. The term library, also referred to herein as a sequencing
library, refers to the collection of nucleic acid fragments from single cells containing known
universal sequences at their 3' and 5' ends. The library includes whole genome nucleic acids
from one or more of the isolated nuclei.
The dual-index fragments can be subjected to conditions that select for a predetermined
size range, such as from 150 to 400 nucleotides in length, such as from 150 to 300 nucleotides.
The resulting dual-index fragments are pooled, and optionally can be subjected to a clean-up
process to enhance the purity to the DNA molecules by removing at least a portion of
unincorporated universal adapters or primers. Any suitable clean-up process may be used, such
as electrophoresis, size exclusion chromatography, or the like. In some embodiments, solid
phase reversible immobilization paramagnetic beads may be employed to separate the desired
DNA molecules from unattached universal adapters or primers, and to select nucleic acids based
on size. Solid phase reversible immobilization paramagnetic beads are commercially available
from Beckman Coulter (Agencourt AMPure XP), Thermofisher (MagJet), Omega Biotek (Mag-
Bind), Promega Beads (Promega), and Kapa Biosystems (Kapa Pure Beads).
The plurality of dual-indexed fragments can be prepared for sequencing. After the dual-
indexed fragments are pooled they are enriched, typically by immobilization and/or
amplification, prior to sequencing ( block 18; block 21). Methods for attaching
dual-indexed fragments from one or more sources to a substrate are known in the art. In one
embodiment, dual-index fragments are enriched using a plurality of capture oligonucleotides
having specificity for the dual-index fragments, and the capture oligonucleotides can be
immobilized on a surface of a solid substrate. For instance, capture oligonucleotides can include
a first member of a universal binding pair, and wherein a second member of the binding pair is
immobilized on a surface of a solid substrate. Likewise, methods for amplifying immobilized
dual-indexed fragments include, but are not limited to, bridge amplification and kinetic
exclusion. Methods for immobilizing and amplifying prior to sequencing are described in, for
instance, Bignell et al. (US 8,053,192), Gunderson et al. (W02016/130704), Shen et al. (US
8,895,249), and Pipenburg et al. (US 9,309,502).
A pooled sample can be immobilized in preparation for sequencing. Sequencing can be
performed as an array of single molecules, or can be amplified prior to sequencing. The
amplification can be carried out using one or more immobilized primers. The immobilized
primer(s) can be, for instance, a lawn on a planar surface, or on a pool of beads. The pool of
beads can be isolated into an emulsion with a single bead in each "compartment" of the
emulsion. At a concentration of only one template per "compartment," only a single template is
amplified on each bead.
The term "solid-phase amplification" as used herein refers to any nucleic acid
amplification reaction carried out on or in association with a solid support such that all or a
portion of the amplified products are immobilized on the solid support as they are formed. In
particular, the term encompasses solid-phase polymerase chain reaction (solid-phase PCR) and
solid phase isothermal amplification which are reactions analogous to standard solution phase
amplification, except that one or both of the forward and reverse amplification primers is/are
immobilized on the solid support. Solid phase PCR covers systems such as emulsions, wherein
one primer is anchored to a bead and the other is in free solution, and colony formation in solid
phase gel matrices wherein one primer is anchored to the surface, and one is in free solution.
In some embodiments, the solid support comprises a patterned surface. A "patterned
surface" refers to an arrangement of different regions in or on an exposed layer of a solid
support. For example, one or more of the regions can be features where one or more
amplification primers are present. The features can be separated by interstitial regions where
amplification primers are not present. In some embodiments, the pattern can be an x-y format of
features that are in rows and columns. In some embodiments, the pattern can be a repeating
arrangement of features and/or interstitial regions. In some embodiments, the pattern can be a
random arrangement of features and/or interstitial regions. Exemplary patterned surfaces that
can be used in the methods and compositions set forth herein are described in US Pat. Nos.
8,778,848, 8,778,849 and 9,079,148, and US Pub. No. 2014/0243224.
In some embodiments, the solid support includes an array of wells or depressions in a
surface. This may be fabricated as is generally known in the art using a variety of techniques,
including, but not limited to, photolithography, stamping techniques, molding techniques and
microetching techniques. As will be appreciated by those in the art, the technique used will
depend on the composition and shape of the array substrate.
The features in a patterned surface can be wells in an array of wells (e.g. microwells or
nanowells) on glass, silicon, plastic or other suitable solid supports with patterned, covalently-
linked gel such as poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM, see,
for example, US Pub. No. 2013/184796, , and ). The process
creates gel pads used for sequencing that can be stable over sequencing runs with a large number
of cycles. The covalent linking of the polymer to the wells is helpful for maintaining the gel in
the structured features throughout the lifetime of the structured substrate during a variety of uses.
However, in many embodiments the gel need not be covalently linked to the wells. For example,
in some conditions silane free acrylamide (SFA, see, for example, US Pat. No. 8,563,477) which
is not covalently attached to any part of the structured substrate, can be used as the gel material.
In particular embodiments, a structured substrate can be made by patterning a solid
support material with wells (e.g. microwells or nanowells), coating the patterned support with a
gel material (e.g. PAZAM, SFA or chemically modified variants thereof, such as the azidolyzed
version of SFA (azido-SFA)) and polishing the gel coated support, for example via chemical or
mechanical polishing, thereby retaining gel in the wells but removing or inactivating
substantially all of the gel from the interstitial regions on the surface of the structured substrate
between the wells. Primer nucleic acids can be attached to gel material. A solution of dual-
index fragments can then be contacted with the polished substrate such that individual dual-index
fragments will seed individual wells via interactions with primers attached to the gel material;
however, the target nucleic acids will not occupy the interstitial regions due to absence or
inactivity of the gel material. Amplification of the dual-index fragments will be confined to the
wells since absence or inactivity of gel in the interstitial regions prevents outward migration of
the growing nucleic acid colony. The process can be conveniently manufactured, being scalable
and utilizing conventional micro- or nanofabrication methods.
Although the disclosure encompasses "solid-phase" amplification methods in which only
one amplification primer is immobilized (the other primer usually being present in free solution),
in one embodiment it is preferred for the solid support to be provided with both the forward and
the reverse primers immobilized. In practice, there will be a 'plurality' of identical forward
primers and/or a 'plurality' of identical reverse primers immobilized on the solid support, since
the amplification process requires an excess of primers to sustain amplification. References
herein to forward and reverse primers are to be interpreted accordingly as encompassing a
'plurality' of such primers unless the context indicates otherwise.
As will be appreciated by the skilled reader, any given amplification reaction requires at
least one type of forward primer and at least one type of reverse primer specific for the template
to be amplified. However, in certain embodiments the forward and reverse primers may include
template-specific portions of identical sequence, and may have entirely identical nucleotide
sequence and structure (including any non-nucleotide modifications). In other words, it is
possible to carry out solid-phase amplification using only one type of primer, and such single-
primer methods are encompassed within the scope of the disclosure. Other embodiments may use
forward and reverse primers which contain identical template-specific sequences but which
differ in some other structural features. For example, one type of primer may contain a non-
nucleotide modification which is not present in the other.
In all embodiments of the disclosure, primers for solid-phase amplification are preferably
immobilized by single point covalent attachment to the solid support at or near the 5' end of the
primer, leaving the template-specific portion of the primer free to anneal to its cognate template
and the 3' hydroxyl group free for primer extension. Any suitable covalent attachment means
known in the art may be used for this purpose. The chosen attachment chemistry will depend on
the nature of the solid support, and any derivatization or functionalization applied to it. The
primer itself may include a moiety, which may be a non-nucleotide chemical modification, to
facilitate attachment. In a particular embodiment, the primer may include a sulphur-containing
nucleophile, such as phosphorothioate or thiophosphate, at the 5' end. In the case of solid-
supported polyacrylamide hydrogels, this nucleophile will bind to a bromoacetamide group
present in the hydrogel. A more particular means of attaching primers and templates to a solid
support is via 5' phosphorothioate attachment to a hydrogel comprised of polymerized
acrylamide and N-(5-bromoacetamidylpentyl) acrylamide (BRAPA), as described in WO
05/065814.
Certain embodiments of the disclosure may make use of solid supports that include an
inert substrate or matrix (e.g. glass slides, polymer beads, etc.) which has been "functionalized,"
for example by application of a layer or coating of an intermediate material including reactive
groups which permit covalent attachment to biomolecules, such as polynucleotides. Examples of
such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert
substrate such as glass. In such embodiments, the biomolecules (e.g. polynucleotides) may be
directly covalently attached to the intermediate material (e.g. the hydrogel), but the intermediate
material may itself be non-covalently attached to the substrate or matrix (e.g. the glass substrate).
The term "covalent attachment to a solid support" is to be interpreted accordingly as
encompassing this type of arrangement.
The pooled samples may be amplified on beads wherein each bead contains a forward
and reverse amplification primer. In a particular embodiment, the library of dual-index fragments
is used to prepare clustered arrays of nucleic acid colonies, analogous to those described in U.S.
Pub. No. 2005/0100900, U.S. Pat. No. 7,115,400, WO 00/18957 and WO 98/44151 by solid-
phase amplification and more particularly solid phase isothermal amplification. The terms
' cluster' and 'colony' are used interchangeably herein to refer to a discrete site on a solid support
including a plurality of identical immobilized nucleic acid strands and a plurality of identical
immobilized complementary nucleic acid strands. The term "clustered array" refers to an array
formed from such clusters or colonies. In this context, the term "array" is not to be understood as
requiring an ordered arrangement of clusters.
The term "solid phase" or "surface" is used to mean either a planar array wherein primers
are attached to a flat surface, for example, glass, silica or plastic microscope slides or similar
flow cell devices; beads, wherein either one or two primers are attached to the beads and the
beads are amplified; or an array of beads on a surface after the beads have been amplified.
Clustered arrays can be prepared using either a process of thermocycling, as described in
WO 98/44151, or a process whereby the temperature is maintained as a constant, and the cycles
of extension and denaturing are performed using changes of reagents. Such isothermal
amplification methods are described in patent application numbers WO 02/46456 and U.S. Pub.
No. 2008/0009420. Due to the lower temperatures useful in the isothermal process, this is
particularly preferred in some embodiments.
It will be appreciated that any of the amplification methodologies described herein or
generally known in the art may be used with universal or target-specific primers to amplify
immobilized DNA fragments. Suitable methods for amplification include, but are not limited to,
the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription
mediated amplification (TMA) and nucleic acid sequence based amplification (NASBA), as
described in U.S. Pat. No. 8,003,354. The above amplification methods may be employed to
amplify one or more nucleic acids of interest. For example, PCR, including multiplex PCR,
SDA, TMA, NASBA and the like may be utilized to amplify immobilized DNA fragments. In
some embodiments, primers directed specifically to the polynucleotide of interest are included in
the amplification reaction.
Other suitable methods for amplification of polynucleotides may include oligonucleotide
extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232
(1998)) and oligonucleotide ligation assay (OLA) (See generally U.S. Pat. Nos. 7,582,420,
,185,243, 5,679,524 and 5,573,907; EP 0 320 308 Bl; EP 0 336 731 Bl; EP 0 439 182 Bl; WO
90/01069; WO 89/12696; and WO 89/09835) technologies. It will be appreciated that these
amplification methodologies may be designed to amplify immobilized DNA fragments. For
example, in some embodiments, the amplification method may include ligation probe
amplification or oligonucleotide ligation assay (OLA) reactions that contain primers directed
specifically to the nucleic acid of interest. In some embodiments, the amplification method may
include a primer extension-ligation reaction that contains primers directed specifically to the
nucleic acid of interest. As a non-limiting example of primer extension and ligation primers that
may be specifically designed to amplify a nucleic acid of interest, the amplification may include
primers used for the GoldenGate assay (Illumina, Inc., San Diego, CA) as exemplified by U.S.
Pat. No. 7,582,420 and 7,611,869.
Exemplary isothermal amplification methods that may be used in a method of the present
disclosure include, but are not limited to, Multiple Displacement Amplification (MDA) as
exemplified by, for example Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002) or
isothermal strand displacement nucleic acid amplification exemplified by, for example U.S. Pat.
No. 6,214,587. Other non-PCR-based methods that may be used in the present disclosure
include, for example, strand displacement amplification (SDA) which is described in, for
example Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995; U.S.
Pat. Nos. 5,455,166, and 5,130,238, and Walker et al., Nucl. Acids Res. 20:1691-96 (1992) or
hyper-branched strand displacement amplification which is described in, for example Lage et al.,
Genome Res. 13:294-307 (2003). Isothermal amplification methods may be used with, for
instance, the strand-displacing Phi 29 polymerase or B st DNA polymerase large fragment, 5'->3'
exo- for random primer amplification of genomic DNA. The use of these polymerases takes
advantage of their high processivity and strand displacing activity. High processivity allows the
polymerases to produce fragments that are 10-20 kb in length. As set forth above, smaller
fragments may be produced under isothermal conditions using polymerases having low
processivity and strand-displacing activity such as Klenow polymerase. Additional description
of amplification reactions, conditions and components are set forth in detail in the disclosure of
U.S. Patent No. 7,670,810.
Another polynucleotide amplification method that is useful in the present disclosure is
Tagged PCR which uses a population of two-domain primers having a constant 5' region
followed by a random 3' region as described, for example, in Grothues et al. Nucleic Acids Res.
21(5):1321-2 (1993). The first rounds of amplification are carried out to allow a multitude of
initiations on heat denatured DNA based on individual hybridization from the randomly-
synthesized 3' region. Due to the nature of the 3' region, the sites of initiation are contemplated to
be random throughout the genome. Thereafter, the unbound primers may be removed and further
replication may take place using primers complementary to the constant 5' region.
In some embodiments, isothermal amplification can be performed using kinetic exclusion
amplification (KEA), also referred to as exclusion amplification (ExAmp). A nucleic acid
library of the present disclosure can be made using a method that includes a step of reacting an
amplification reagent to produce a plurality of amplification sites that each includes a
substantially clonal population of amplicons from an individual target nucleic acid that has
seeded the site. In some embodiments, the amplification reaction proceeds until a sufficient
number of amplicons are generated to fill the capacity of the respective amplification site. Filling
an already seeded site to capacity in this way inhibits target nucleic acids from landing and
amplifying at the site thereby producing a clonal population of amplicons at the site. In some
embodiments, apparent clonality can be achieved even if an amplification site is not filled to
capacity prior to a second target nucleic acid arriving at the site. Under some conditions,
amplification of a first target nucleic acid can proceed to a point that a sufficient number of
copies are made to effectively outcompete or overwhelm production of copies from a second
target nucleic acid that is transported to the site. For example, in an embodiment that uses a
bridge amplification process on a circular feature that is smaller than 500 nm in diameter, it has
been determined that after 14 cycles of exponential amplification for a first target nucleic acid,
contamination from a second target nucleic acid at the same site will produce an insufficient
number of contaminating amplicons to adversely impact sequencing-by-synthesis analysis on an
Il lumina sequencing platform.
In some embodiments, amplification sites in an array can be, but need not be, entirely
clonal. Rather, for some applications, an individual amplification site can be predominantly
populated with amplicons from a first dual-indexed fragment and can also have a low level of
contaminating amplicons from a second target nucleic acid. An array can have one or more
amplification sites that have a low level of contaminating amplicons so long as the level of
contamination does not have an unacceptable impact on a subsequent use of the array. For
example, when the array is to be used in a detection application, an acceptable level of
contamination would be a level that does not impact signal to noise or resolution of the detection
technique in an unacceptable way. Accordingly, apparent clonality will generally be relevant to a
particular use or application of an array made by the methods set forth herein. Exemplary levels
of contamination that can be acceptable at an individual amplification site for particular
applications include, but are not limited to, at most 0.1%, 0.5%, 1%, 5%, 10% or 25%
contaminating amplicons. An array can include one or more amplification sites having these
exemplary levels of contaminating amplicons. For example, up to 5%, 10%, 25%, 50%, 75%, or
even 100% of the amplification sites in an array can have some contaminating amplicons. It will
be understood that in an array or other collection of sites, at least 50%, 75%, 80%, 85%, 90%,
95% or 99% or more of the sites can be clonal or apparently clonal.
In some embodiments, kinetic exclusion can occur when a process occurs at a sufficiently
rapid rate to effectively exclude another event or process from occurring. Take for example the
making of a nucleic acid array where sites of the array are randomly seeded with dual-indexed
fragments from a solution and copies of the dual-indexed fragments are generated in an
amplification process to fill each of the seeded sites to capacity. In accordance with the kinetic
exclusion methods of the present disclosure, the seeding and amplification processes can proceed
simultaneously under conditions where the amplification rate exceeds the seeding rate. As such,
the relatively rapid rate at which copies are made at a site that has been seeded by a first target
nucleic acid will effectively exclude a second nucleic acid from seeding the site for
amplification. Kinetic exclusion amplification methods can be performed as described in detail
in the disclosure of US Application Pub. No. 2013/0338042.
Kinetic exclusion can exploit a relatively slow rate for initiating amplification (e.g. a
slow rate of making a first copy of a dual-index fragment) vs. a relatively rapid rate for making
subsequent copies of the dual-indexed fragment (or of the first copy of the dual-indexed
fragment). In the example of the previous paragraph, kinetic exclusion occurs due to the
relatively slow rate of dual-indexed fragment seeding (e.g. relatively slow diffusion or transport)
vs. the relatively rapid rate at which amplification occurs to fill the site with copies of the dual-
indexed fragment seed. In another exemplary embodiment, kinetic exclusion can occur due to a
delay in the formation of a first copy of a dual-indexed fragment that has seeded a site (e.g.
delayed or slow activation) vs. the relatively rapid rate at which subsequent copies are made to
fill the site. In this example, an individual site may have been seeded with several different dual-
indexed fragments (e.g. several dual-indexed fragments can be present at each site prior to
amplification). However, first copy formation for any given dual-indexed fragment can be
activated randomly such that the average rate of first copy formation is relatively slow compared
to the rate at which subsequent copies are generated. In this case, although an individual site
may have been seeded with several different dual-indexed fragments, kinetic exclusion will
allow only one of those dual-indexed fragments to be amplified. More specifically, once a first
dual-indexed fragment has been activated for amplification, the site will rapidly fill to capacity
with its copies, thereby preventing copies of a second dual-indexed fragment from being made at
the site.
In one embodiment, the method is carried out to simultaneously (i) dual-index fragments
to amplification sites at an average transport rate, and (ii) amplify the dual-index fragments that
are at the amplification sites at an average amplification rate, wherein the average amplification
rate exceeds the average transport rate (U.S. Pat. No. 9,169,513). Accordingly, kinetic exclusion
can be achieved in such embodiments by using a relatively slow rate of transport. For example, a
sufficiently low concentration of dual-index fragments can be selected to achieve a desired
average transport rate, lower concentrations resulting in slower average rates of transport.
Alternatively or additionally, a high viscosity solution and/or presence of molecular crowding
reagents in the solution can be used to reduce transport rates. Examples of useful molecular
crowding reagents include, but are not limited to, polyethylene glycol (PEG), ficoll, dextran, or
polyvinyl alcohol. Exemplary molecular crowding reagents and formulations are set forth in U.S.
Pat. No. 7,399,590, which is incorporated herein by reference. Another factor that can be
adjusted to achieve a desired transport rate is the average size of the target nucleic acids.
An amplification reagent can include further components that facilitate amplicon
formation and in some cases increase the rate of amplicon formation. An example is a
recombinase. Recombinase can facilitate amplicon formation by allowing repeated
invasion/extension. More specifically, recombinase can facilitate invasion of a dual-index
fragment by the polymerase and extension of a primer by the polymerase using the dual-indexed
fragment as a template for amplicon formation. This process can be repeated as a chain reaction
where amplicons produced from each round of invasion/extension serve as templates in a
subsequent round. The process can occur more rapidly than standard PCR since a denaturation
cycle (e.g. via heating or chemical denaturation) is not required. As such, recombinase-
facilitated amplification can be carried out isothermally. It is generally desirable to include ATP,
or other nucleotides (or in some cases non-hydrolyzable analogs thereof) in a recombinase-
facilitated amplification reagent to facilitate amplification. A mixture of recombinase and single
stranded binding (SSB) protein is particularly useful as SSB can further facilitate amplification.
Exemplary formulations for recombinase-facilitated amplification include those sold
commercially as TwistAmp kits by TwistDx (Cambridge, UK). Useful components of
recombinase-facilitated amplification reagent and reaction conditions are set forth in US
,223,414 and US 7,399,590.
Another example of a component that can be included in an amplification reagent to
facilitate amplicon formation and in some cases to increase the rate of amplicon formation is a
helicase. Helicase can facilitate amplicon formation by allowing a chain reaction of amplicon
formation. The process can occur more rapidly than standard PCR since a denaturation cycle
(e.g. via heating or chemical denaturation) is not required. As such, helicase-facilitated
amplification can be carried out isothermally. A mixture of helicase and single stranded binding
(SSB) protein is particularly useful as SSB can further facilitate amplification. Exemplary
formulations for helicase-facilitated amplification include those sold commercially as IsoAmp
kits from Biohelix (Beverly, MA). Further, examples of useful formulations that include a
helicase protein are described in US 7,399,590 and US 7,829,284.
Yet another example of a component that can be included in an amplification reagent to
facilitate amplicon formation and in some cases increase the rate of amplicon formation is an
origin binding protein.
Following attachment of dual-indexed fragments to a surface, the sequence of the
immobilized and amplified dual-indexed fragments is determined. Sequencing can be carried
out using any suitable sequencing technique, and methods for determining the sequence of
immobilized and amplified dual-indexed fragments, including strand re-synthesis, are known in
the art and are described in, for instance, Bignell et al. (US 8,053,192), Gunderson et al.
(W02016/130704), Shen et al. (US 8,895,249), and Pipenburg et al. (US 9,309,502).
The methods described herein can be used in conjunction with a variety of nucleic acid
sequencing techniques. Particularly applicable techniques are those wherein nucleic acids are
attached at fixed locations in an array such that their relative positions do not change and
wherein the array is repeatedly imaged. Embodiments in which images are obtained in different
color channels, for example, coinciding with different labels used to distinguish one nucleotide
base type from another are particularly applicable. In some embodiments, the process to
determine the nucleotide sequence of a dual-index fragment can be an automated process.
Preferred embodiments include sequencing-by-synthesis ("SBS") techniques.
SBS techniques generally involve the enzymatic extension of a nascent nucleic acid
strand through the iterative addition of nucleotides against a template strand. In traditional
methods of SBS, a single nucleotide monomer may be provided to a target nucleotide in the
presence of a polymerase in each delivery. However, in the methods described herein, more than
one type of nucleotide monomer can be provided to a target nucleic acid in the presence of a
polymerase in a delivery.
In one embodiment, a nucleotide monomer includes locked nucleic acids (LNAs) or
bridged nucleic acids (BNAs). The use of LNAs or BNAs in a nucleotide monomer increases
hybridization strength between a nucleotide monomer and a sequencing primer sequence present
on an immobilized dual-index fragment.
SBS can use nucleotide monomers that have a terminator moiety or those that lack any
terminator moieties. Methods using nucleotide monomers lacking terminators include, for
example, pyrosequencing and sequencing using y-phosphate-labeled nucleotides, as set forth in
further detail herein. In methods using nucleotide monomers lacking terminators, the number of
nucleotides added in each cycle is generally variable and dependent upon the template sequence
and the mode of nucleotide delivery. For SBS techniques that utilize nucleotide monomers
having a terminator moiety, the terminator can be effectively irreversible under the sequencing
conditions used as is the case for traditional Sanger sequencing which utilizes
dideoxynucleotides, or the terminator can be reversible as is the case for sequencing methods
developed by Solexa (now Il lumina, Inc.).
SBS techniques can use nucleotide monomers that have a label moiety or those that lack
a label moiety. Accordingly, incorporation events can be detected based on a characteristic of the
label, such as fluorescence of the label; a characteristic of the nucleotide monomer such as
molecular weight or charge; a byproduct of incorporation of the nucleotide, such as release of
pyrophosphate; or the like. In embodiments where two or more different nucleotides are present
in a sequencing reagent, the different nucleotides can be distinguishable from each other, or
alternatively the two or more different labels can be the indistinguishable under the detection
techniques being used. For example, the different nucleotides present in a sequencing reagent
can have different labels and they can be distinguished using appropriate optics as exemplified
by the sequencing methods developed by Solexa (now Illumina, Inc.).
Preferred embodiments include pyrosequencing techniques. Pyrosequencing detects the
release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the
nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996)
"Real-time DNA sequencing using detection of pyrophosphate release." Analytical Biochemistry
242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing." Genome
Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) "A sequencing method based on
real-time pyrophosphate." Science 281(5375), 363; U.S. Pat. Nos. 6,210,891; 6,258,568 and
6,274,320). In pyrosequencing, released PPi can be detected by being immediately converted to
adenosine triphosphate (ATP) by ATP sulfurase, and the level of ATP generated is detected via
luciferase-produced photons. The nucleic acids to be sequenced can be attached to features in an
array and the array can be imaged to capture the chemiluminescent signals that are produced due
to incorporation of a nucleotides at the features of the array. An image can be obtained after the
array is treated with a particular nucleotide type (e.g. A, T, C or G). Images obtained after
addition of each nucleotide type will differ with regard to which features in the array are
detected. These differences in the image reflect the different sequence content of the features on
the array. However, the relative locations of each feature will remain unchanged in the images.
The images can be stored, processed and analyzed using the methods set forth herein. For
example, images obtained after treatment of the array with each different nucleotide type can be
handled in the same way as exemplified herein for images obtained from different detection
channels for reversible terminator-based sequencing methods.
In another exemplary type of SBS, cycle sequencing is accomplished by stepwise
addition of reversible terminator nucleotides containing, for example, a cleavable or
photobleachable dye label as described, for example, in WO 04/018497 and U.S. Pat. No.
7,057,026. This approach is being commercialized by Solexa (now Illumina Inc.), and is also
described in WO 91/06678 and WO 07/123,744. The availability of fluorescently-labeled
terminators in which both the termination can be reversed and the fluorescent label cleaved
facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-
engineered to efficiently incorporate and extend from these modified nucleotides.
In some reversible terminator-based sequencing embodiments, the labels do not
substantially inhibit extension under SBS reaction conditions. However, the detection labels can
be removable, for example, by cleavage or degradation. Images can be captured following
incorporation of labels into arrayed nucleic acid features. In particular embodiments, each cycle
involves simultaneous delivery of four different nucleotide types to the array and each nucleotide
type has a spectrally distinct label. Four images can then be obtained, each using a detection
channel that is selective for one of the four different labels. Alternatively, different nucleotide
types can be added sequentially and an image of the array can be obtained between each addition
step. In such embodiments, each image will show nucleic acid features that have incorporated
nucleotides of a particular type. Different features will be present or absent in the different
images due the different sequence content of each feature. However, the relative position of the
features will remain unchanged in the images. Images obtained from such reversible terminator-
SBS methods can be stored, processed and analyzed as set forth herein. Following the image
capture step, labels can be removed and reversible terminator moieties can be removed for
subsequent cycles of nucleotide addition and detection. Removal of the labels after they have
been detected in a particular cycle and prior to a subsequent cycle can provide the advantage of
reducing background signal and crosstalk between cycles. Examples of useful labels and removal
methods are set forth herein.
In particular embodiments some or all of the nucleotide monomers can include reversible
terminators. In such embodiments, reversible terminators/cleavable fluorophores can include
fluorophores linked to the ribose moiety via a 3' ester linkage (Metzker, Genome Res. 15:1767-
1776 (2005)). Other approaches have separated the terminator chemistry from the cleavage of the
fluorescence label (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005)). Ruparel et al.
described the development of reversible terminators that used a small 3' allyl group to block
extension, but could easily be deblocked by a short treatment with a palladium catalyst. The
fluorophore was attached to the base via a photocleavable linker that could easily be cleaved by a
second exposure to long wavelength UV light. Thus, either disulfide reduction or
photocleavage can be used as a cleavable linker. Another approach to reversible termination is
the use of natural termination that ensues after placement of a bulky dye on a dNTP. The
presence of a charged bulky dye on the dNTP can act as an effective terminator through steric
and/or electrostatic hindrance. The presence of one incorporation event prevents further
incorporations unless the dye is removed. Cleavage of the dye removes the fluorophore and
effectively reverses the termination. Examples of modified nucleotides are also described in U.S.
Pat. Nos. 7,427,673, and 7,057,026.
Additional exemplary SBS systems and methods which can be utilized with the methods
and systems described herein are described in U.S. Pub. Nos. 2007/0166705, 2006/0188901,
2006/0240439, 2006/0281109, 2012/0270305, and 2013/0260372, U.S. Pat. No. 7,057,026, PCT
Publication No. WO 05/065814, U.S. Patent Application Publication No. 2005/0100900, and
PCT Publication Nos. WO 06/064199 and WO 07/010,251.
Some embodiments can use detection of four different nucleotides using fewer than four
different labels. For example, SBS can be performed using methods and systems described in
the incorporated materials of U.S. Pub. No. 2013/0079232. As a first example, a pair of
nucleotide types can be detected at the same wavelength, but distinguished based on a difference
in intensity for one member of the pair compared to the other, or based on a change to one
member of the pair (e.g. via chemical modification, photochemical modification or physical
modification) that causes apparent signal to appear or disappear compared to the signal detected
for the other member of the pair. As a second example, three of four different nucleotide types
can be detected under particular conditions while a fourth nucleotide type lacks a label that is
detectable under those conditions, or is minimally detected under those conditions (e.g., minimal
detection due to background fluorescence, etc.). Incorporation of the first three nucleotide types
into a nucleic acid can be determined based on presence of their respective signals and
incorporation of the fourth nucleotide type into the nucleic acid can be determined based on
absence or minimal detection of any signal. As a third example, one nucleotide type can include
label(s) that are detected in two different channels, whereas other nucleotide types are detected in
no more than one of the channels. The aforementioned three exemplary configurations are not
considered mutually exclusive and can be used in various combinations. An exemplary
embodiment that combines all three examples, is a fluorescent-based SBS method that uses a
first nucleotide type that is detected in a first channel (e.g. dATP having a label that is detected in
the first channel when excited by a first excitation wavelength), a second nucleotide type that is
detected in a second channel (e.g. dCTP having a label that is detected in the second channel
when excited by a second excitation wavelength), a third nucleotide type that is detected in both
the first and the second channel (e.g. dTTP having at least one label that is detected in both
channels when excited by the first and/or second excitation wavelength) and a fourth nucleotide
type that lacks a label that is not, or minimally, detected in either channel (e.g. dGTP having no
label).
Further, as described in the incorporated materials of U.S. Pub. No. 2013/0079232,
sequencing data can be obtained using a single channel. In such so-called one-dye sequencing
approaches, the first nucleotide type is labeled but the label is removed after the first image is
generated, and the second nucleotide type is labeled only after a first image is generated. The
third nucleotide type retains its label in both the first and second images, and the fourth
nucleotide type remains unlabeled in both images.
Some embodiments can use sequencing by ligation techniques. Such techniques use DNA
ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides.
The oligonucleotides typically have different labels that are correlated with the identity of a
particular nucleotide in a sequence to which the oligonucleotides hybridize. As with other SBS
methods, images can be obtained following treatment of an array of nucleic acid features with
the labeled sequencing reagents. Each image will show nucleic acid features that have
incorporated labels of a particular type. Different features will be present or absent in the
different images due the different sequence content of each feature, but the relative position of
the features will remain unchanged in the images. Images obtained from ligation-based
sequencing methods can be stored, processed and analyzed as set forth herein. Exemplary SBS
systems and methods which can be utilized with the methods and systems described herein are
described in U.S. Pat. Nos. 6,969,488, 6,172,218, and 6,306,597.
Some embodiments can use nanopore sequencing (Deamer, D. W. & Akeson, M.
"Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18, 147-
151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore
analysis", Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J.
A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope"
Nat. Mater. 2:611-615 (2003)). In such embodiments, the dual-index fragment passes through a
nanopore. The nanopore can be a synthetic pore or biological membrane protein, such as a-
hemolysin. As the dual-index fragment passes through the nanopore, each base-pair can be
identified by measuring fluctuations in the electrical conductance of the pore. (U.S. Pat. No.
7,001,792; Soni, G. V. & Meller, "A. Progress toward ultrafast DNA sequencing using solid-
state nanopores." Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based single-
molecule DNA analysis." Nanomed. 2, 459-481 (2007); Cockroft, S. L., Chu, J., Amorin, M. &
Ghadiri, M. R. "A single-molecule nanopore device detects DNA polymerase activity with
single-nucleotide resolution." J. Am. Chem. Soc. 130, 818-820 (2008)). Data obtained from
nanopore sequencing can be stored, processed and analyzed as set forth herein. In particular, the
data can be treated as an image in accordance with the exemplary treatment of optical images
and other images that is set forth herein.
Some embodiments can use methods involving the real-time monitoring of DNA
polymerase activity. Nucleotide incorporations can be detected through fluorescence resonance
energy transfer (FRET) interactions between a fluorophore-bearing polymerase and y-phosphate-
labeled nucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and 7,211,414, or
nucleotide incorporations can be detected with zero-mode waveguides as described, for example,
in U.S. Pat. No. 7,315,019, and using fluorescent nucleotide analogs and engineered polymerases
as described, for example, in U.S. Pat. No. 7,405,281 and U.S. Pub. No. 2008/0108082. The
illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase
such that incorporation of fluorescently labeled nucleotides can be observed with low
background (Levene, M. J. et al. "Zero-mode waveguides for single-molecule analysis at high
concentrations." Science 299, 682-686 (2003); Lundquist, P. M. et al. "Parallel confocal
detection of single molecules in real time." Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al.
"Selective aluminum passivation for targeted immobilization of single DNA polymerase
molecules in zero-mode waveguide nano structures." Proc. Natl. Acad. Sci. USA 105, 1176-1181
(2008)). Images obtained from such methods can be stored, processed and analyzed as set forth
herein.
Some SBS embodiments include detection of a proton released upon incorporation of a
nucleotide into an extension product. For example, sequencing based on detection of released
protons can use an electrical detector and associated techniques that are commercially available
from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and
systems described in U.S. Pub. Nos. 2009/0026082; 2009/0127589; 2010/0137143; and
2010/0282617. Methods set forth herein for amplifying target nucleic acids using kinetic
exclusion can be readily applied to substrates used for detecting protons. More specifically,
methods set forth herein can be used to produce clonal populations of amplicons that are used to
detect protons.
The above SBS methods can be advantageously carried out in multiplex formats such that
multiple different dual-index fragments are manipulated simultaneously. In particular
embodiments, different dual-index fragments can be treated in a common reaction vessel or on a
surface of a particular substrate. This allows convenient delivery of sequencing reagents,
removal of unreacted reagents and detection of incorporation events in a multiplex manner. In
embodiments using surface-bound target nucleic acids, the dual-index fragments can be in an
array format. In an array format, the dual-index fragments can be typically bound to a surface in
a spatially distinguishable manner. The dual-index fragments can be bound by direct covalent
attachment, attachment to a bead or other particle or binding to a polymerase or other molecule
that is attached to the surface. The array can include a single copy of a dual-index fragment at
each site (also referred to as a feature) or multiple copies having the same sequence can be
present at each site or feature. Multiple copies can be produced by amplification methods such
as, bridge amplification or emulsion PCR as described in further detail herein.
The methods set forth herein can use arrays having features at any of a variety of
densities including, for example, at least about 10 features/cm2, 100 features/ cm2, 500 features/
cm2, 1,000 features/ cm2, 5,000 features/ cm2, 10,000 features/ cm2, 50,000 features/ cm2,
100,000 features/ cm2, 1,000,000 features/ cm2, 5,000,000 features/ cm2, or higher.
An advantage of the methods set forth herein is that they provide for rapid and efficient
detection of a plurality of cm2, in parallel. Accordingly, the present disclosure provides
integrated systems capable of preparing and detecting nucleic acids using techniques known in
the art such as those exemplified herein. Thus, an integrated system of the present disclosure can
include fluidic components capable of delivering amplification reagents and/or sequencing
reagents to one or more immobilized dual-index fragments, the system including components
such as pumps, valves, reservoirs, fluidic lines and the like. A flow cell can be configured and/or
used in an integrated system for detection of target nucleic acids. Exemplary flow cells are
described, for example, in U.S. Pub. No. 2010/0111768 and US Ser. No. 13/273,666. As
exemplified for flow cells, one or more of the fluidic components of an integrated system can be
used for an amplification method and for a detection method. Taking a nucleic acid sequencing
embodiment as an example, one or more of the fluidic components of an integrated system can
be used for an amplification method set forth herein and for the delivery of sequencing reagents
in a sequencing method such as those exemplified above. Alternatively, an integrated system
can include separate fluidic systems to carry out amplification methods and to carry out detection
methods. Examples of integrated sequencing systems that are capable of creating amplified
nucleic acids and also determining the sequence of the nucleic acids include, without limitation,
the MiSeqTM platform (Illumina, Inc., San Diego, CA) and devices described in US Ser. No.
13/273,666.
Also provided herein are compositions. During the practice of the methods described
herein various compositions can result. For example, a composition including chemically treated
nucleosome-depleted isolated nuclei, where isolated nuclei include indexed nucleic acid
fragments, can result. Also provided is a multi-well plate, wherein a well of the multi-well plate
includes isolated nuclei having indexed nucleic acid fragments. In one embodiment, isolated
nuclei can include non-natural cross-links, such as the type of cross-links formed by a cross-
linking agent, e.g., formaldehyde. In one embodiment, indexed nucleic acid fragments terminate
in a cleaved restriction site having an overhang. In one embodiment, the isolated nuclei
comprise rearranged genomic DNA.
EMBODIMENTS
Embodiment 1.
A method of preparing a sequencing library comprising nucleic acids from
a plurality of single cells, the method comprising:
(a) providing isolated nuclei from a plurality of cells;
(b) subjecting the isolated nuclei to a chemical treatment to generate nucleosome-
depleted nuclei, while maintaining integrity of the isolated nuclei;
(c) distributing subsets of the nucleosome-depleted nuclei into a first plurality of
compartments and contacting each subset with a transposome complex, wherein the transposome
complex in each compartment comprises a transposase and a first index sequence that is different
from first index sequences in the other compartments;
(d) fragmenting nucleic acids in the subsets of nucleosome-depleted nuclei into a
plurality of nucleic acid fragments and incorporating the first index sequences into at least one
strand of the nucleic acid fragments to generate indexed nuclei comprising indexed nucleic acid
fragments, wherein the indexed nucleic acid fragments remain attached to the transposases;
(e) combining the indexed nuclei to generate pooled indexed nuclei;
distributing subsets of the pooled indexed nuclei into a second plurality of
compartments;
incorporating into the indexed nucleic acid fragments in each compartment a
second index sequence to generate dual-index fragments, wherein the second index sequence in
each compartment is different from second index sequences in the other compartments;
combining the dual-index fragments, thereby producing a sequencing library
comprising whole genome nucleic acids from the plurality of single cells.
Embodiment 2. The method of Embodiment 1, wherein the chemical treatment comprises
a treatment with a chaotropic agent capable of disrupting nucleic acid-protein interactions.
Embodiment 3. The method of Embodiment 2 or 3, wherein the chaotropic agent
comprises lithium 3,5-diiodosalicylic acid.
Embodiment 4. The method of any of Embodiments 1 to 3, wherein the chemical
treatment comprises a treatment with a detergent capable of disrupting nucleic acid-protein
interactions.
Embodiment 5. The method of any of Embodiments 1 to 4, wherein the detergent
comprises sodium dodecyl sulfate (SDS).
Embodiment 6. The method of any of Embodiments 1 to 5, wherein the nuclei are treated
with a cross-linking agent prior to step (b).
Embodiment 7. The method of any of Embodiments 1 to 6, wherein the cross-linking
agent is formaldehyde.
Embodiment 8. The method of any of Embodiments 1 to 7, wherein the concentration of
formaldehyde ranges from about 0.2% to about 2%.
Embodiment 9.
The method of any of Embodiments 1 to 8, wherein the concentration of
formaldehyde is no greater than about 1.5%.
Embodiment 10. The method of any of Embodiments 1 to 9, wherein the cross-linking by
formaldehyde is reversed after step (f) and prior to step (g).
Embodiment 11.
The method of any of Embodiments 1 to 10, wherein the reversal of the
cross-linking comprises incubation at about 55°C to about 72°C.
Embodiment 12. The method of any of Embodiments 1 to 11, wherein the transposases are
disassociated from the indexed nucleic acid fragments prior to the reversal of the cross-linking.
Embodiment 13. The method of any of Embodiments 1 to 12, wherein the transposases are
disassociated from the indexed nucleic acid fragments using sodium dodecyl sulfate (SDS).
Embodiment 14. The method of any of Embodiments 1 to 13, wherein the nuclei are treated
with a restriction enzyme prior to step (d).
Embodiment 15.
The method of any of Embodiments 1 to 14, wherein the nuclei are treated
with a ligase after treatment with the restriction enzyme.
Embodiment 16.
The method of any of Embodiments 1 to 15, wherein the distributing in
steps (c) and (f) is performed by fluorescence-activated nuclei sorting.
Embodiment 17. The method of any of Embodiments 1 to 16, wherein the subsets of the
nucleosome-depleted nuclei comprise approximately equal numbers of nuclei.
Embodiment 18. The method of any of Embodiments 1 to 17, wherein the subsets of the
nucleosome-depleted nuclei comprise from 1 to about 2000 nuclei.
Embodiment 19. The method of any of Embodiments 1 to 18, wherein the first plurality of
compartments is a multi-well plate.
Embodiment 20. The method of any of Embodiments 1 to 19, wherein the multi-well plate
is a 96-well plate or a 384-well plate.
Embodiment 21.
The method of any of Embodiments 1 to 20, wherein the subsets of the
pooled indexed nuclei comprise approximately equal numbers of nuclei.
Embodiment 22. The method of any of Embodiments 1 to 21, wherein the subsets of the
pooled indexed nuclei comprise from 1 to about 25 nuclei.
Embodiment 23. The method of any of Embodiments 1 to 22, wherein the subsets of the
pooled indexed nuclei include at least 10 times fewer nuclei than the subsets of the nucleosome-
depleted nuclei.
Embodiment 24. The method of any of Embodiments 1 to 23, wherein the subsets of the
pooled indexed nuclei include at least 100 times fewer nuclei than the subsets of the nucleosome-
depleted nuclei.
Embodiment 25. The method of any of Embodiments 1 to 24, wherein the second plurality
of compartments is a multi-well plate.
Embodiment 26. The method of any of Embodiments 1 to 25, wherein the multi-well plate
is a 96-well plate or a 384-well plate.
Embodiment 27. The method of any of Embodiments 1 to 26, wherein step (c) comprises
adding the transposome complex to the compartments after the subsets of nucleosome-depleted
nuclei are distributed.
Embodiment 28.
The method of any of Embodiments 1 to 27, wherein each of the
transposome complexes comprises a transposon, each of the transposons comprising a
transferred strand.
Embodiment 29.
The method of any of Embodiments 1 to 28, wherein the transferred strand
comprises the first index sequence and a first universal sequence.
Embodiment 30. The method of any of Embodiments 1 to 29, wherein the incorporation of
the second index sequence in step (g) comprises contacting the indexed nucleic acid fragments in
each compartment with a first universal primer and a second universal primer, each comprising
an index sequence and each comprising a sequence identical to or complementary to a portion of
the first universal sequence, and performing an exponential amplification reaction.
Embodiment 31.
The method of any of Embodiments 1 to 30, wherein the index sequence
of the first universal primer is the reverse complement of the index sequence of the second
universal primer.
Embodiment 32.
The method of any of Embodiments 1 to 31, wherein the index sequence
of the first universal primer is different from the reverse complement of the index sequence of
the second universal primer.
Embodiment 33. The method of any of Embodiments 1 to 32, wherein the first universal
primer further comprises a first capture sequence and a first anchor sequence complementary to a
universal sequence at the 3' end of the dual-index fragments.
Embodiment 34. The method of any of Embodiments 1 to 33, wherein the first capture
sequence comprises the P5 primer sequence.
Embodiment 35. The method of any of Embodiments 1 to 34, wherein the second universal
primer further comprises a second capture sequence and a second anchor sequence
complementary to a universal sequence at the 5' end of the dual-index fragments.
Embodiment 36. The method of any of Embodiments 1 to 35, wherein the second capture
sequence comprises the reverse complement of the P7 primer sequence.
Embodiment 37.
The method of any of Embodiments 1 to 36, wherein the exponential
amplification reaction comprises a polymerase chain reaction (PCR).
Embodiment 38. The method of any of Embodiments 1 to 37, wherein the PCR comprises
15 to 30 cycles.
Embodiment 39. The method of any of Embodiments 1 to 38, further comprising an
enrichment of dual-index fragments using a plurality of capture oligonucleotides having
specificity for the dual-index fragments.
Embodiment 40. The method of any of Embodiments 1 to 39, wherein the capture
oligonucleotides are immobilized on a surface of a solid substrate.
Embodiment 41.
The method of any of Embodiments 1 to 40, wherein the capture
oligonucleotides comprise a first member of a universal binding pair, and wherein a second
member of the binding pair is immobilized on a surface of a solid substrate.
Embodiment 42.
The method of any of Embodiments 1 to 42, further comprising
sequencing of the dual-index fragments to determine the nucleotide sequence of nucleic acids
from the plurality of single cells.
Embodiment 43. The method of any of Embodiments 1 to 42, further comprising:
providing a surface comprising a plurality of amplification sites, wherein the
amplification sites comprise at least two populations of attached single stranded capture
oligonucleotides having a free 3' end, and
contacting the surface comprising amplification sites with the dual-index fragments under
conditions suitable to produce a plurality of amplification sites that each comprise a clonal
population of amplicons from an individual dual-index fragment.
Embodiment 44. The method of any of Embodiments 1 to 43, wherein the number of the
dual-index fragments exceeds the number of amplification sites, wherein the dual-index
fragments have fluidic access to the amplification sites, and wherein each of the amplification
sites comprises a capacity for several dual-index fragments in the sequencing library.
Embodiment 45.
The method of any of Embodiments 1 to 44, wherein the contacting
comprises simultaneously (i) transporting the dual-index fragments to the amplification sites at
an average transport rate, and (ii) amplifying the dual-index fragments that are at the
amplification sites at an average amplification rate, wherein the average amplification rate
exceeds the average transport rate.
Embodiment 46. A composition comprising chemically treated nucleosome-depleted
isolated nuclei, wherein the isolated nuclei comprise indexed nucleic acid fragments.
Embodiment 47.
The composition of Embodiment 46, wherein the isolated nuclei comprise
non-natural cross-links.
Embodiment 48. The composition of any of Embodiments 46 or 47, wherein the
composition comprises indexed nucleic acid fragments that terminate in a cleaved restriction site
comprising an overhang.
Embodiment 49. The composition of any of Embodiments 46 to 48, wherein the isolated
nuclei comprise rearranged genomic DNA.
Embodiment 50. A multi-well plate, wherein a well of the multi-well plate comprises the
composition of any of Embodiments 46-49.
The present disclosure is illustrated by the following examples. It is to be understood that
the particular examples, materials, amounts, and procedures are to be interpreted broadly in
accordance with the scope and spirit of the disclosure as set forth herein.
EXAMPLE 1
Generating and sequencing thousands of single-cell genomes with combinatorial indexing
Single-cell genome sequencing has proven valuable for the detection of somatic
variation, particularly in the context of tumor evolution. Current technologies suffer from high
library construction costs which restrict the number of cells that can be assessed and thus impose
limitations on the ability to measure heterogeneity within a tissue. Here, Single cell
Combinatorial Indexed Sequencing (SCI-seq) is presented as a way of simultaneously generating
thousands of low-pass single cell libraries for somatic copy number variant detection. Libraries
for 16,698 single cells were constructed from a combination of cultured cell lines, primate frontal
cortex tissue, and two human adenocarcinomas, including a detailed assessment of subclonal
variation within a pancreatic tumor. This Example is also available as Vitak et al. (2017, Nature
Methods, 14, 302 308, doi.10.1038/maiedi 4154)
Methods
Sample preparation and nuclei isolation.
Tissue culture cell lines were trypsinized then pelleted if adherent (He La S3, ATCC
CCL-2.2; NIH/3T3, ATCC CRL-1658) or pelleted if grown in suspension (GM12878, Coriell;
karyotyped at the OHSU Research Cytogenetics Laboratory), followed by one wash with ice
cold PBS. They were then carried through crosslinking (for the xSDS method) or directly into
nuclei preparation using Nuclei Isolation Buffer (NIB, 10 mM TrisHC1 pH7.4, 10 mM NaC1, 3
mM MgC12, 0.1% Igepal ®, lx protease inhibitors (Roche, Cat. 11873580001)) with or without
nucleosome depletion. Tissue samples (RhesusFcxl, RhesusFcx2, PDAC, CRC) were dounce
homogenized in NIB then passed through a 351.tm cell strainer prior to nucleosome depletion.
The frozen Rhesus frontal cortex samples, RhesusFcxl (4 yr. female) and RhesusFcx2 (9 yr.
female), were obtained from the Oregon National Primate Research Center as a part of their
aging nonhuman primate resource.
Standard Single Cell Library Construction
Single cell libraries constructed using quasi-random priming (QRP) and degenerate
oligonucleotide primed PCR (DOP) were prepared from isolated nuclei without nucleosome
depletion and brought up to 1 mL of NIB, stained with 5 [IL of 5 mg/ml DAPI (Thermo Fisher,
Cat. D1306) then FANS sorted on a Sony SH800 in single cell mode. One nucleus was deposited
into each single well containing the respective sample buffers. QRP libraries were prepared using
the PicoPlex DNA-seq Kit (Rubicon Genomics, Cat. R300381) according to the manufacturer's
protocol and using the indexed PCR primers provided in the kit. DOP libraries were prepared
using the SeqPlex DNA Amplification Kit (Sigma, Cat. SEQXE-50RXN) according to the
manufacturer's protocol, but with the use of custom PCR indexing primers that contain 10 by
index sequences. To avoid over-amplification, all QRP and DOP libraries were amplified with
the addition of 0.5 !IL of 100X SYBR Green (FMC BioProducts, Cat. 50513) on a BioRad CFX
thermocycler in order to monitor the amplification and pull reactions that have reached mid-
exponential amplification.
Nucleosome Depletion
Lithium assisted nucleosome depletion (LAND): Prepared Nuclei were pelleted and
resuspended in NIB supplemented with 200 [EL of 12.5 mM lithium 3,5-diiodosalicylic acid
(referred to as Lithium diiodosalicylate in main text, Sigma, Cat. D3635) for 5 minutes on ice
prior to the addition of 800 [EL NIB and then taken directly into flow sorting.
Cross linking and SDS nucleosome depletion (xSDS): Cross linking was achieved by
incubating cells in 10 mL of media (cell culture) or nuclei in 10 mL of HEPES NIB (20 mM
HEPES, 10 mM NaC1, 3mM MgC12, 0.1% igepal, lx protease inhibitors (Roche, Cat.
11873580001)) (tissue samples) containing 1.5% formaldehyde at room for 10 minutes. The
crosslinking reaction was neutralized by bringing the reaction to 200 mM Glycine (Sigma, Cat.
G8898-500G) and incubating on ice for 5 minutes. Cell culture samples were crosslinked and
then washed once with 10 ml ice cold lx PBS and had nuclei isolated by incubating in NIB
buffer on ice for 20 minutes and pelleted once again. Nuclei were then resuspended in 800 uL lx
NEBuffer 2.1 (NEB, Cat. B7202S) with 0.3% SDS (Sigma, Cat. L3771) and incubated at 42°C
with vigorous shaking for 30 minutes in a thermomixer (Eppendorf). SDS was then quenched by
the addition of 200 [EL of 10% Triton-X100 (Sigma, Cat. 90021) and incubated at 42°C with
vigorous shaking for 30 minutes.
Combinatorial indexing via tagmentation and PCR
Nuclei were stained with 5 [EL of 5mg/m1DAPI (Thermo Fisher, Cat. D1306) and passed
through a 35 [tm cell strainer. A 96 well plate was prepared with 10 [EL of lx Nextera® Tagment
DNA (TD) buffer from the Nextera® DNA Sample Preparation Kit (Illumina, Cat. FC1031)
diluted with NIB in each well. A Sony SH800 flow sorter was used to sort 2,000 single nuclei
into each well of the 96 well tagmentation plate in fast sort mode. Next, 1 [EL of a uniquely
indexed 2.5 [tM transposase-adaptor complex (transposome) was added to each well. These
complexes and associated sequences are described in Amini et. al. (Amini, S. et al. Nat. Genet.
46, 1343-9, 2014). Reactions were incubated at 55°C for 15 minutes. After cooling to room
temperature, all wells were pooled and stained with DAPI as previously described. A second 96
well plate, or set of 96 well plates, were prepared with each well containing 8.5 [EL of a 0.058%
SDS, 8.9 nM BSA solution and 2.5 pL of 2 uniquely barcoded primers at 10 p.M. 22 post-
tagmentation nuclei from the pool of 96 reactions were then flow sorted on the same instrument
but in single cell sort mode into each well of the second plate and then incubated in the SDS
solution at 55°C for 5 minutes to disrupt the nuclear scaffold and disassociate the transposase
enzyme. Crosslinks were reversed by incubating at 68°C for an hour (xSDS). SDS was then
diluted by the addition of 7.5 [IL of Nextera® PCR Master mix (Illumina, Cat. FC1031) as
well as 0.5 [IL of 100X SYBR Green (FMC BioProducts, Cat. 50513) and 4 [IL of water. Real
time PCR was then performed on a BioRad CFX thermocycler by first incubating reactions at
72°C for 5 minutes, prior to 3 minutes at 98°C and 15-20 cycles of [20 sec. at 98°C, 15 sec. at
63°C, and 25 sec. at 72°C]. Reactions were monitored and stopped once exponential
amplification was observed in a majority of wells. 5 [IL of each well was then pooled and
purified using a Qiaquick PCR Purification column (Qiagen, Cat. 28104) and eluted in 30 [IL of
EB.
Library quantification and sequencing
Libraries were quantified between the range of 200bp and 1 kbp on a High Sensitivity
Bioanalyzer kit (Agilent, Cat. 5067-4626). Libraries were sequenced on an Illumina NextSeq®
500 loaded at 0.8 pM with a custom sequencing chemistry protocol (Read 1: 50 imaged cycles;
Index Read 1: 8 imaged cycles, 27 dark cycles, 10 imaged cycles; Index Read 2: 8 imaged
cycles, 21 dark cycles, 10 imaged cycles; Read 2: 50 imaged cycles) using custom sequencing
primers described in Amini et. al. (Amini, S. et al. Nat. Genet. 46, 1343-9, 2014). QRP and DOP
libraries were sequenced using standard primers on the NextSeq® 500 using high-capacity 75
cycle kits with dual-indexing. For QRP there is an additional challenge that the first 15 by of the
read are highly enriched for "G" bases, which are non-fluorescent with the NextSeq® 2-color
chemistry and therefore cluster identification on the instrument fails. The libraries were therefore
sequenced using a custom sequencing protocol that skips this region (Read 1: 15 dark cycles, 50
imaged cycles; Index Read 1: 10 imaged cycles; Index Read 2: 10 imaged cycles).
Sequence Read Processing
Software for processing SCI-seq raw reads is available on the World Wide Web at sci-
seq.sourceforge.net. Sequence runs were processed using bcl2fastq (Illumina Inc., version
2.15.0) with the --create-fastq-for-index-reads and --with-failed-reads options to produce fastq
files. Index reads were concatenated (36 by total) and used as the read name with a unique read
number appended to the end. These indexes were then matched to the corresponding index
reference sets allowing for a hamming distance of two for each of the four index components (i7-
Transposase (8 bp), i7-PCR (10 bp), i5-Transposase (8 bp), and i5-PCR (10 bp)), reads matching
a quad-index combination were then renamed to the exact index (and retained the unique read
number) which was subsequently used as the cell identifier. Reads were then adaptor trimmed,
then paired and unpaired reads were aligned to reference genomes by Bowtie2 and merged.
Human preparations were aligned to GRCh37, Rhesus preparations were aligned to RheMac8,
and Human/Mouse mix preparations were aligned to a combined human (GRCh37) and mouse
(mm10) reference. Aligned bam files were subjected to PCR duplicate removal using a custom
script that removes reads with identical alignment coordinates on a per-barcode basis along with
reads with an alignment score less than 10 as reported by Bowtie2.
Single Cell Discrimination
For each PCR plate, a total of 9,216 unique index combinations are possible (12 i7-
Transposase indexes x 8 i5-Transposase indexes x 12 i7-PCR indexes X 8 i5-PCR indexes), for
which only a minority should have a substantial read count, as the majority of index
combinations should be absent - i.e. transposase index combinations of nuclei that were not
sorted into a given PCR well. These "empty" indexes typically contain very few reads (1-3% of a
run) with the majority of reads falling into bona fide single cell index combinations (97-99% of a
run). The resulting histogram of logio unique read counts for index combinations (
produces a mix of two normal distributions: a noise component and a single cell component. The
R package "mixtools" was then used to fit a mixed model (normalmixEM) to identify the
proportion (X) mean (1,t) and standard deviation (a) of each component. The read count threshold
to qualify as a single cell library was taken to be the greater of either one standard deviation
below the mean of the single cell component in logio space, or 100 fold greater than the mean of
the noise component (+2 in logio space), and had to be a minimum of 1,000 unique reads.
Human-Mouse Mix Experiments
One of two approaches was taken to mix human (GM12878 or HeLa S3) and mouse
(3T3) cells: i) mixing at the cell stage (HumMus.LAND1 and HumMus.LAND2) or ii) mixing at
the nuclei stage (HumMus.LAND3, HumMus.LAND4, and HumMus.xSDS). The latter was
employed to control for nuclei crosslinking or agglomerating together that could result in
doublets. Libraries were constructed as described herein, for instances where two distinct DAPI-
positive populations were observed during flow sorting, included both populations in the same
gate so as not to skew proportions. Reads were processed as in other experiments, except reads
were instead aligned to a reference comprised of GRCh37 (hg19) and mm10. The mapping
quality 10 filter effectively removed reads that aligned to conserved regions in both genomes and
then for each identified single cell, reads to each species were tallied and used to estimate
collision frequency. For early LAND preparations 25 indexed nuclei were sorted per PCR well
and produced total collision rates (i.e. twice the human-mouse collision rate) of 28.1% and
.4%. For the second two LAND preparations we sorted 22 nuclei per PCR well, which
produced a total collision rate of 4.3% for one preparation and no detectable collisions in
another. We also tested two FANS sorting conditions for our xSDS preparation, one was
permissive and allowed a broader range of DAPI fluorescence, and the other more restrictive,
and carried out both preparations on separate sides of the same PCR plate. For the permissive
gating we observed a total collision rate of 23.6% with a substantial reduction for the more
restrictive gating at 8.1%. Based on these results we decided to continue sorting 22 nuclei per
PCR well using the more restrictive FANS
Library Depth Projections
To estimate the performance of a library pool if, or when, it was sequenced to a greater
depth, random reads were incrementally sampled from each SCI-seq preparation across all index
combinations including unaligned and low quality reads without replacement at every one
percent of the total raw reads. For each point we identified the total number reads that are aligned
with high quality (MQ > 10) assigned to each single cell index and the fraction of those reads
that are unique, non-PCR duplicates, as well as the corresponding fraction of total reads sampled
that were assigned to that index. Using these points we fit both a nonlinear model and a Hanes-
Woolfe transformed model to predict additional sequencing for each individual single cell library
within the pool and projected out to a median unique read percentage across cells of5%. To
determine the accuracy of the models, we determined the number of downsampled raw reads of
each library that would reach the point in which the median unique read percentage per cell was
90%, which is somewhat less than what was achieved for libraries that were sequenced at low
coverage. We then subsampled the pre-determined number of reads for 30 iterations and built a
new model for each cell at each iteration and then predicted the unique read counts for each cell
out to the true sequencing depth that was achieved. The standard deviation of the true read count
across all iterations for all cells was then calculated.
Genome Windowing
Genomic windows were determined on a per-library basis using custom tools. For each
chromosome the size of the entire chromosome was divided by the target window size to
produce the number of windows per chromosome. The total read count for the chromosome
summarized over the pool of all single cells (GM12878 for all human samples where absolute
copy number was determined, as well as for each pooled sample where amplifications or
deletions relative to the mean copy number were determined) was then divided by the window
count to determine the mean read count per window. The chromosome was then walked and
aligned reads from the pool tallied and a window break was made once the target read count per
window was reached. Windows at chromosome boundaries were only included if they contained
more than 75% of the average reads per window limit for that chromosome. By using dynamic
windows we accounted for biases, such as highly repetitive regions, centromeres and other
complex regions that can lead to read dropout in the case of fixed size bins22.
GC Bias Correction
Reads were placed into the variable sized bins and GC corrected based on individual read
GC content instead of the GC content of the dynamic windows. We posit that the large bin sizes
needed for single cell analysis average out smaller scale GC content changes. Furthermore, SCI-
seq does not involve pre-amplification where large regions of the genome are amplified,
therefore GC bias originates solely from the PCR and is amplicon-specific. To calculate
correction weights for the reads we compared the fraction of all reads with a given GC to the
fraction of total simulated reads with the average insert size at the same GC fraction. This weight
was then used in lieu of read counts and summed across all reads in a given window. All regions
present in DAC blacklisted regions were excluded from analysis for the human sample analyses
(http://genome.ucsc.edu/cgi-bin/hgFileUi?db=hg19&g=wgEncodeMapability)19. Following GC
correction, all reads were normalized by the average number of reads per bin across the genome.
Finally, for each window we took the normalized read count of each cell and divided it by the
pooled sample baseline to produce a ratio score.
Measures of data variation
To measure data quality, we calculated two different measures of coverage dispersion:
the median absolute deviation (MAD), the median absolute pairwise difference (MAPD). For
each score we calculated the median of the absolute values of all pairwise differences between
neighboring bins that have been normalized by the mean bin count within the cell (log2-
normalized ratios for the MAPD scores). These scores measure the dispersion of normalized
binned reads due to technical noise, rather than due copy number state changes, which are less
frequent2'22.
Copy Number Variant Calling
CNV calling was performed on the windowed, GC corrected and bulk sample normalized
reads with two available R packages that employ two different segmentation strategies: a Hidden
Markov Model approach (HMMcopy, version 3.3.0, Ha, G. et al., Genome Res. 22, 1995-2007,
2012) and Circular Binary Segmentation (DNAcopy, version 1.44.0, Olshen et al. Biostatistics 5,
557-572, 2004). Values were Loge transformed for input (2*log2 for CBS) and copy number
calls were made based on the optimized parameters from Knouse et al. 2016, Knouse et al.,
Genome Res. gr.198937.115, 2016, doi:10.1101/gr.198937.115). For optimal sensitivity and
specificity to detect copy number calls with sizes >5Mb we set the probability of segment
extension (E) to 0.995 for EIMM and for CBS we chose the significance level to accept a copy
number change (a) to be 0.0001. The Loge cutoffs for calling losses or gains were 0.4 and -0.35
for HMIM and 1.32 and 0.6 for CBS. As an additional tool for CNV calling we used Ginkgo22,
which uses an alternative method for data normalization. We uploaded bed files for each cell and
a bulk down sampled bed file, which we created with Picard Tools (we used a down sample
probability of 0.1). For the analysis we chose to segment single cells with the down sampled bulk
bed file and when ploidy was known for the samples we created FACS files to force Ginkgo to
normalize to that ploidy. Calls for the three methods were intersected either on a per-window
basis or were filtered to only include calls that span > 80% of a chromosome arm and then
intersected for aneuploidy analysis.
Tumor breakpoint analysis
Unlike the assessment of sporadic aneuploidy, tumor structural variation is much more
complex with a large portion of breakpoints within chromosomes. Further, sporadic aneuploidy
within any given subclone of a tumor is less pertinent than an accurate profile of the
subpopulations that are present. We therefore used the HMIM and CBS segmented ratio score
matrixes to identify breakpoints by tallying up the boundaries of segmented regions across cells.
We then used the resulting distribution of shared chromosomal breakpoints across the genome to
identify local maxima to account for variability in which specific window the call was made, and
then retained those that are present in at least 5% of cells. We then merged all windows within
each breakpoint span and calculated the new log2 ratio of each aneuploid cell over the mean
values of the euploid population. We then carried out principle components analysis prior to k-
means clustering with a k value determined by Silhouette analysis. To minimize the effect of
doublets which can account for -10% of putative single cells and also to exclude low-
performance cells, we retained only those in the close proximity to their respective centroids. We
then merged sequence reads for all cells within each cluster and then carried out a higher
resolution CNV analysis (target window size of 100 kbp) using an HMM strategy followed by
absolute copy number state identification and the identification of focal amplifications and
deletions using a sliding window outlier strategy20. Intra-tumoral clonal relationships are most
accurately captured by shared breakpoints as opposed to the drift in copy number of a segment
based on the assumption that structural changes involving breaks in the DNA as being more
impactful on the cell. We therefore compared cells by assessing the proportion of segments
between breakpoints that were identified using the high resolution (100 kbp) CNV analysis that
overlapped by at least 90% (to account for noise in the exact window that was called as the copy
number change) out of the total number of segments.
Results
Nucleosome depletion for uniform genome coverage
A hurdle to adapt combinatorial indexing to produce uniformly distributed sequence
reads is the removal of nucleosomes bound to genomic DNA without compromising nuclear
integrity. The sciATAC-seq method is carried out on native chromatin, which permits the
conversion of DNA into library molecules only within regions of open chromatin (1-4% of the
genome)'8. This restriction is desirable for epigenetic characterization; however, for CNV
detection, it results in biological bias and severely limited read counts (3,000 per cell)''. We
therefore developed two strategies to unbind nucleosomes from genomic DNA while retaining
nuclear integrity for SCI-seq library construction. The first, Lithium Assisted Nucleosome
Depletion (LAND), utilizes the chaotropic agent, Lithium diiodosalycylate, to disrupt DNA-
protein interactions in the cell, therefore releasing DNA from histones. The second, crosslinking
with SDS (xSDS), uses the detergent SDS to denature histone proteins and render them unable to
bind DNA. However, SDS has a disruptive effect on nuclear integrity, thus necessitating a
crosslinking step prior to denaturation in order to maintain intact nuclei.
To test the viability of these strategies, we performed bulk (30,000 nuclei) preparations
on the HeLa S3 cell line, for which chromatin accessibility and genome structure has been
extensively profiled19'20, and carried out LAND or xSDS treatments along with a standard
control. In all three cases, nuclei remained intact -a key requirement for the SCI-seq workflow
(). Prepared nuclei were then carried through standard ATAC-seq library construction'.
The library prepared from untreated nuclei produced the expected ATAC-seq signal with a 10.8
fold enrichment of sequence reads aligning to annotated HeLa S3 accessibility sites. Both the
LAND and xSDS preparations had substantially lower enrichments of 2.8 and 2.2 fold
respectively, close to the 1.4 fold observed for shotgun sequencing (, Table 1).
Furthermore, the projected number of unique sequence reads present in the LAND and xSDS
preparations were 1.7 billion and 798 million respectively, much greater than for the standard
library at 170 million, suggesting a larger proportion of the genome was converted into viable
sequencing molecules.
Table 1. Bulk library statistics. Information on bulk cell libraries constructed to evaluate
nucleosome depletion. *SHOT library is a random sampling of 60M reads obtained from the
He La dbGaP repository under accession: phs000640.v4.pl (The ENCODE Project
Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489,
57-74 (2012). Library size estimates were generated using Picard tools function
"EstimateLibraryComplexity". For shotgun sequencing, the read used were duplicate removed,
and therefore duplication rate and library size estimates were not determined.
Reads in Percent
Bulk Duplication Fold Estimated
Reads MCW 0 He La DHS in DHS
Library Rate Enrichment Library Size
sites s ites
xSDS 4.50% 83,507,827 2,307,825 2.76% 2.22 798,085,544
LAND 1.86% 64,353,617 2,240,466 3.48% 2.79 1,657.844,868
ATAC 27.24% 60,494,125 8,179,083 13.52% 10.84 170 409,197
SHOT* NA 60,000,000 1,031,310 1.72% 1.38 NA
SCI-seq with nucleosome depletion
To assess the performance of nucleosome depletion with our single cell combinatorial
indexing workflow, we first focused on the deeply profiled, euploid lymphoblastoid cell line
GM1287814'15'19. We produced a total of six SCI-seq libraries with a variety of LAND
conditions, each using a single 96-well plate at the PCR indexing stage, and a single xSDS
library with 3 x 96-well PCR plates. To serve as a comparison to existing methods, we prepared
42 single cell libraries using quasi-random priming (QRP, 40 passing QC) and 51 using
degenerate oligonucleotide primed PCR (DOP, 45 passing QC). Finally, we karyotyped 50 cells
to serve as a non-sequencing means of aneuploidy measurement (Table 2).
Table 2a. EM Mixed Model
Nucleosome PCR Nuc.
Library Sample A (noise, single cell) p (noise, single cell)
Wells / well
Depletion Method
GM12878.LAND1 96 25 0.66137
Human (GM12878) LAND (27.6 pM LIS) 0.872007,0.127993 1.080594,3.841161
GM12878.LAND2 96 25 0.513341
Human (GM12878) 0.419749,0.580251 0.291373,1.982663
LAND (13.8 pM LIS)
LAND (13.8 pM LIS
GM12878.NSTLAND 96 22 0.736942
Human (GM12878) 0.752279,0.247721 1.177030,3.937951
+ 200 mM NaCI)
x -link + LAND (13.8
GM12878 xLAND 96 22
Human (GM12878) 0.803801,0.196199 0.814446,3.409897 0.578019
pM LIS)
GM12878.LAND3 96 22 0.680607
Human (GM12878) 0.842110,0.157890 1.307204,4.047124
LAND (13.8 pM LIS)
GM12878.LAND4 96 22
Human (GM12878) 0.861427,0.138573 1.184529,3.689950 0.619864
LAND (4.6 pM LIS)
Arrested, LAND (4.6
GM12878.arrLAND 96 22 1.280424,4.3764043 0.526405
Human (GM12878) 0.970847,0.0291532
pM LIS)
HeLa.LAND1 96 22 1.489698,4.622590 0.740663
Human (He La S3) LAND (4.6 pM LIS) 0.884456,0.115544
HeLa.LAND2 96 22 0.496199
Human (He La S3) LAND (4.6 pM LIS) 0.849262,0.150738 0.816437,3.448150
HeLa.LAND3 96 22
Human (He La S3) LAND (4.6 pM LIS) 0.838170,0.161830 1.476571,4.135318 0.539156
Human (He La S3),
HumMus.LAND1 96 25 0.559918
LAND (27.6 pM LIS) 0.816623,0.183377 0.826636,2.662703
Mouse (3T3)
Human (He La S3),
LAND (113.8 pM
HumMus.LAND2 96 25 0.784437,0.215563 1.223024,3.960925 0.716764
Mouse (3T3)
LIS)
Human (GM12878),
HumMus.LAND3 96 22 0.627049
LAND (4.6 pM LIS) 0.863399,0.136601 1.473206,4.590961
Mouse (3T3)
Human (GM12878),
HumMus LAND4 96 22
0.973846,0.0261538 1.448882,5.0360715 0.712699
LAND (4.6 pM LIS)
Mouse (3T3)
Rhesus Individual 1
RhesusInd1. LAND 16 22 1.09558
0.823777,0.176223 1.774362,4.301835
LAND (4.6 pM LIS)
(frozen)
GM12878.xSDS xSDS 288 22
Human (GM12878) 0.871926,0.128074 1.781897,4.291739 0.764169
Human (He La S3),
HumMus.xSDS xSDS 96 22 0.878084
0.868349,0.131651 1.776006,4.209856
Mouse (3T3)
Stage 2 Colorectal
CRC xSDS xSDS 16 22 0.83885
0.911423,0.0885767 1.423343,4.7335258
Cancer (frozen)
Stage 3 Pancreatic
PDAC.xSDS Ductal Adenocarcinoma xSDS 288 22 1.713041,4.4984682 0.872799
0.915855,0.0841453
(fresh)
Rhesus Individual 1
RhesusInc11.xSDS xSDS 96 22
0.953348,0.0466516 1.122175,4.4798411 0.788582
(frozen)
Rhesus Individual 2
RhesusInd2.xSDS xSDS 96 22 1.091425,4.5055530 0.763775
0.931090,0.0689105
(frozen)
Table 2b.
Sequenced Reads
Median Cells 5e4
Single Cell Single Cell Median Unique Mean Unique
Library
Read Cutoff Libraries MCW 0 Reads MCW 0 Reads Reads
Complexity
GM12878.LAND1 1,512 621 11,721 37,055 45.96 129
GM12878.LAND2 113 90.79 0
1,000 2,091 3,434
GM12878.NSTLAND 71.33 313
1,588 1,060 13,734 52,244
GM12878.xLAND 58.80 72
1,000 1,212 6,384 14,148
GM12878.LAND3 34.42 232
2,325 1,015 16,673 84,010
GM12878.LAND4 616 87.55 68
1,529 7,151 32,614
GM12878.arrLAND 119 36.51 54
7,079 33,923 94,036
HeLa.LAND1 573 91.01 338
7,619 67,077 100,016
HeLa.LAND2 648 37.45 29
1,000 4,756 18,026
HeLa.LAND3 97.73 120
3,946 1,140 18,695 25,501
HumMus.LAND1 263 2.90 4
1,000 2,699 6,174
HumMus.LAND2 71.31 388
1,754 1,346 13,876 51,952
HumMus.LAND3 9.202 645 61.408 74.329 96.69 378
HumMus.LAND4 21,055 115 119,428 359,175 95.84 99
RhesusInd1.LAND 5,947 340 141,449 165,453 88.21 248
GM12878.xSDS 6,051 3,123 29,550 64,986 53.08 1,056
HumMus.xSDS 87.89 605
,970 1,331 44,699 64,659
CRC.xSDS 151 89.70 111
7,846 72,753 110,823
PDAC.xSDS 68.60 846
,164 1,715 49,272 86,592
RhesusInd1.xSDS 171 24.36 92
4,912 55,142 120,769
Rhesuslnd2.xSDS 381 23.76 213
,517 62,731 122,602
16,698 5,395
Table 2. SCI-seq library summary. Information on library construction and statistics on the actual depth obtained for each SCI-seq library
preparation. (a) Details of library construction and the mixed model used to determine the read count threshold for single cell libraries. (b)
Details on libraries for the actual sequence depth obtained in this study.
For each SCI-seq preparation, the number of potential index combinations is 96
(transposase indexing) x N (PCR indexing, 96 per plate); however, not all index combinations
represent a single cell library, as each PCR well contains only 15-25 transposase-indexed nuclei.
To identify non-empty index combinations, we generated a logio transformed histogram of
unique (i.e. non-PCR duplicate), high-quality (MQ > 10) aligned reads for each potential index
combination. This resulted in a bimodal distribution comprised of a low-read-count, noise
component centered between 50 and 200 reads, and a high-read-count, single cell component
centered between 10,000 and 100,000 reads (FIG 7a,b, . We then used a mixed model to
identify indexes that fall in this high-read-count component (, which resulted in 4,643
single cell libraries across the six SCI-seq preparations that used LAND for nucleosome
depletion and 3,123 for the xSDS preparation.
To confirm that the majority of putative single cell libraries contain true single cells, we
carried out four SCI-seq library preparations on a mix of human and mouse cells using LAND
(2,369 total cells) with either 22 or 25 nuclei per PCR well, and one preparation using xSDS split
between two FANS conditions (1,367 total cells; . For each experiment we analyzed the
proportion of putative single cells with > 90% of their reads that aligned exclusively to the
human or mouse genome. The remaining cells represent human-mouse collisions (i.e. doublets)
and make up approximately half of the total collision rate (the remaining half being human-
human or mouse-mouse). The total collision rates varied between 0-23.6%, and were used to
decide upon 22 nuclei per well with restrictive sorting conditions for a target doublet frequency
of <10%, comparable to sciATAC-seq17 or high throughout single cell RNA-seq technologies21.
The unique read count produced for each library in a SCI-seq preparation is a function of
library complexity and sequencing depth. Due to the inhibitive cost of deeply sequencing every
preparation during development, we implemented a model to project the anticipated read count
and PCR duplicate percentage that would be achieved with increased sequencing depth (,
Methods). As a means of quality assessment, we identified the depth at which a median of 50%
of reads across cells are PCR duplicates (M50), representing the point at which additional
sequencing becomes excessive (i.e. greater than 50% of additional reads provide no new
information), along with several other metrics (Table 3). Model projections from a subset of the
sequenced reads accurately predicted the actual median unique read count within a median of
0.02% (maximum 2.25%, mean 0.41%) across all libraries. As further confirmation, additional
sequencing of a subset of PCR wells from several preparations produced unique reads counts for
each cell that were within a median of 0.13% (maximum 3.56%, mean 0.72%) of what was
predicted by our model ().
Table 3a.
Library Projections
Projected to Median of 50% Projected to Median of 25% Projected to Median of 10%
Complexity Complexity Complexity
Median Mean Raw Reads Median Mean Raw Reads Median Mean Raw Reads
Library
GM12878.LAND1 378.305 1.176.230 1.640.000.000 554.638 1.734.782 4.850.000.000 653.064 2.115.189 14.120.000.000
GM12878.NSTLAND
44,608 155,477 350,000,000 68,418 228,101 1,030,000,000 83,359 269,951 2,980,000,000
GM12878.LAND3
218,132 1,318,212 2,430,000,000 323,878 2,133,376 7,220,000,000 399,607 2,718,203 21,050,000,000
GM12878.LAND4
135,114 746,530 810,000,000 200,422 1,239,687 2,390,000,000 246,204 2,082,902 6,940,000,000
GM12878.arrLAND
1,490,817 3,684,539 910,000,000 2,141,086 5,687,397 2,710.000.000 2.792,344 7,024,499 7,900 000,000
He La.LAND1
3,997,311 5,642,469 7,180,000,000 6,140,962 8,861,587 21,380,000,000 7,399,793 11,190,892 62,310,000,000
HeLa.LAND3
736,813 901,740 2,350,000,000 1,107,204 1,415,747 6,970,000,000 1,337,941 1,806,880 20,320,000,000
HumMus.LAND1
,991 79,857 50,000,000 56,355 126,673 150,000,000 70,808 161,428 420,000,000
HumMus.LAND2
44,393 154,148 440,000,000 67,957 226,139 1,290,000,000 82,696 277,161 3,760,000,000
HumMus.LAND3
2,257,543 2,638,358 3,890,000,000 3,453,346 3,957,131 11,600,000,000 4,186,331 4,806,388 33,740,000,000
HumMus.LAND4
4,305,319 11,479,621 3,260,000,000 6,126,707 16,880,151 9,710,000,000 7,474,417 20,732,681 28,290,000,000
RhesusInd1.LAND
454,681 514,445 530,000,000 685,354 756,326 1,570,000,000 823,686 902,566 4,570,000,000
GM12878.xSDS
26,791 63,223 580,000,000 39,666 94,153 1,710,000,000 48,089 113,352 4,980,000,000
CRC.xSDS
352,168 530,978 190,000,000 532,772 790,798 560,000,000 641,639 946,770 1,620,000,000
PDAC.xSDS
71,378 129,304 590,000,000 107,615 191,011 1,750,000,000 130,444 228,852 5,110,000,000
Number of Cells That Can Reach N Reads from Projections
Table 3b.
.00E+04 1.00E+05 1.50E+05 2.50E+05 5.00E+05 7.50E+05 1.00E+06
Library
GM12878.LAND1 619 604 579 504 373 308 268
183 112 78
GM12878.NSTLAND 662 504 439 340
990 886 810 674 470 370 310
GM12878.LAND3
GM12878.LAND4 574 474 403 319 211 167 137
115 107 102
GM12878.arrLAND 119 119 118 117
573 573 573 572 557 547 541
HeLa.LAND1
HeLa.LAND3 941 812
1,140 1,138 1,129 1,115 1,057
HumMus.LAND1 167 113 76 40 19 11 6
HumMus.LAND2 851 636 550 421 228 137 100
634 610 593
HumMus.LAND3 645 645 645 641
115 115 115 115 115 115 115
HumMus.LAND4
Rhesuslndl.LAND 328 299 277 260 219 186 148
183 69 22
GM12878.xSDS 1.804 1.094 769 468
151 147 144 137 107 70 43
CRC.xSDS
PDAC.xSDS 874 601 242 98 54
1,356 1,080
Table 3. SCI-seq library projection statistics. Information on projected statistics of each SCI-seq library if increased
sequencing depth were obtained. Projections use the model described in the methods section. Libraries that either failed
(GM12878.LAND2 and HeLa.LAND2), or were sequenced to saturation for which the projections do not apply
(Rhesus.Indl.xSDS and Rhesus.Ind2.xSDS) are not included. (a) Projections out to a given median complexity including
the raw read count to reach that point. (b) The number of single cells meeting various read count thresholds are listed if
libraries were sequenced to saturation (median complexity of 5%).
Coverage uniformity was assessed using mean absolute deviation (MAD)22 and mean
absolute pairwise deviation (MAPD)2, which indicated substantially better uniformity using
xSDS over LAND (MAD: mean 1.57-fold improvement, p = <1x1015; MAPD: 1.70-fold
improvement, p = <1x10-15, Welch's t-test). The deviation using xSDS is similar to multiple
displacement amplification methods, though still greater than for QRP and DOP ()22.
While LAND preparations had higher coverage bias, they also produced higher unique read
counts per cell (e.g. M50 of 763,813 for one of three HeLa LAND preparations) when compared
to xSDS (e.g. M50 of 63,223 for the GM12878 preparation). For all libraries, we observed the
characteristic 9 basepair overlap of adjacent read pairs due to the mechanism of transposition13'23,
indicating we are able to sequence molecules on either side of a transposase insertion event ().
Copy number variant calling using SCI-seq
For any single cell genome sequencing study, determining how to filter out failed
libraries without removing true aneuploid cells is a significant challenge. We initially proceeded
with CNV calling on our SCI-seq preparations without any filtering in order to directly compare
with other methods. For all preparations, we used cells with a minimum of 50,000 unique, high
quality aligned reads (868 across all LAND libraries, 1,056 for the xSDS library), applied
Ginkgo22, Circular Binary Segmentation (CBS)24, and a Hidden Markov Model (HMM)25, with
variable-sized genomic windows (target median of 2.5 million bp) for CNV calling () and
conservatively retained the intersection of all three methods. To compare our sequencing-based
calls with karyotyped cells, we focused on chromosome-arm level events (,f). Consistent
with the coverage uniformity differences, our LAND SCI-seq preparations produced a high
aneuploidy rate (61.9%), suggesting an abundance of false positives due to lack of coverage
uniformity (,g). However, the xSDS nucleosome depletion strategy with SCI-seq resulted
in an aneuploidy frequency of 22.6%, much closer to the karyotyping results (,h) as well
as DOP and QRP (15.0% and 13.5%, respectively) ().
We next determined filtering criteria based on MAD and MAPD scores across a variety
of resolutions and read count thresholds (). This analysis revealed a greater range of
variability in the resolution of our SCI-seq preparations, which is largely driven by the wider
range of unique reads per cell when compared to standard methods. By applying a MAD
variance filter of 0.2 across all methods, aneuploidy rates for xSDS, DOP and QRP dropped to
12.2%, 9.7% and 10.5% respectively, all below the rate determined by karyotyping, yet closer to
one another than prior to filtering ().
Copy number variation in the Rhesus brain
Estimates of aneuploidy and large-scale CNV frequencies in the mammalian brain vary
widely, from <5% to 33%1-4. This uncertainty largely stems from the inability to profile
sufficient numbers of single cells to produce quantitative measurements. The Rhesus macaque is
an ideal model for quantifying the abundance of aneuploidy in the brain, as human samples are
challenging to acquire and are confounded by high variability in lifetime environmental
exposures. Furthermore, the Rhesus brain is phylogenetically, structurally and physiologically
more similar to humans than rodents'.
To demonstrate the versatility of our platform, we applied LAND and xSDS SCI-seq to
archived frontal cortex tissue (Individual 1), along with 38 cells using QRP (35 passing QC), and
cells using DOP (30 passing QC). Our low-capacity LAND preparation (16 PCR indexes)
produced 340 single cell libraries with a median unique read count of 141,449 (248 cells >
50,000 unique reads), and our xSDS preparation generated 171 single cell libraries with a median
unique read count of 55,142 (92 cells > 50,000 unique reads). The number of cells produced in
our xSDS preparation was lower than expected, largely due to nuclei aggregates during sorting
that may be remedied by additional cell dis-aggregation steps.
Across all methods of library construction we observed greater discrepancies between the
three CNV calling approaches than in the human analyses (-19), likely due to the lower
quality of the Rhesus reference genome (284,705 contigs < 1 Mbp), emphasizing the need for
"platinum" quality reference genomes27. We therefore focused on the HMM results for sub-
chromosomal calls (a) and performed aneuploidy analysis using the intersection of CBS
and HMM calls. Consistent with our cell line results, the LAND preparation produced a much
higher aneuploidy rate (95.1%), suggestive of false positives stemming from coverage
nonuniformity (-22). The xSDS SCI-seq unfiltered aneuploidy rate (25.0%) was close to
the DOP preparation (18.5%), with QRP producing a much lower rate (3.1%; b). After
imposing a variance filter for cells with a MAD score of 0.2 or lower, the aneuploidy rates
dropped to 12.0% for the xSDS preparation, 8.7% for the DOP, and stayed the same for the QRP
preparation at 3.1%. These rates were similar to those produced by xSDS SCI-seq on a 200 mm3
section of frontal cortex from a second individual (381 single cells, median read count of 62,731,
213 cells > 50,000 unique reads) which produced unfiltered and filtered aneuploidy rates of
12.1% and 10.3% respectively ().
SCI-seq on primary tumor samples reveals clonal populations
One of the primary applications of single cell genome sequencing is in the profiling of
tumor heterogeneity and understanding clonal evolution in cancer as it relates to treatment
resistance5-8. We carried out a single xSDS SCI-seq preparation on a freshly acquired stage III
pancreatic ductal adenocarcinoma (PDAC) sample measuring approximately 250 mm3 which
resulted in 1,715 single cell libraries sequenced to a median unique read count of 49,272 per cell
(M50 of 71,378; 846 cells > 50,000 unique reads at the depth the library was sequenced; a). We first performed CNV calling using our GM12878 library as a euploid baseline for
comparison to identify a set of high-confidence euploid cells (298, 35.2%) which were then used
as a new baseline specific to the individual and preparation (, 25, 26). Assuming that
subchromosomal copy number alterations (caused by genome instability) are more informative
for identifying subclonal populations than whole chromosome aneuploidy (due to errors during
cell division), we developed a strategy to identify putative copy number breakpoints at low
resolution to be used as new window boundaries (Methods, ) followed by stratification
via principle components analysis (PCA) and k-means clustering. We initially applied this
method to our HeLa libraries (2,361 single cells in total), revealing no distinct heterogeneity and
further supporting the stability of the HeLa cell line20 (-31), and then on our primary
PDAC sample, which revealed an optimum cluster count of 4 by silhouette analysis (b,c).
The first of these clusters (k3) is a population of euploid cells that were not considered
high confidence euploid in the initial analysis, and thus not removed. When including these, the
euploid population rises to 389 for a final tumor cell purity of 46.0%, within the expected range
for PDAC28. For the remaining clusters kl (199 cells), k2 (115 cells) and k4 (91 cells), we
aggregated all reads from cells proximal to each centroid (Methods) and carried out CNV calling
using 100 kbp windows, a 25-fold greater resolution than the initial analysis, and then
determined absolute copy number states' (d).
Across the three tumor clusters, a substantial portion of copy number segments were
shared (44.8%), suggesting that they arose from a common progenitor population. This includes
a highly rearranged chromosome 19 which harbors a focal amplification of CEBPA, which
encodes an enhancer binding protein, at copy number 7 which is frequently mutated in AML29,
and has recently been shown to have altered epigenetic regulation in pancreatic tumors' (e). An all-by-all pairwise comparison revealed clusters k2 and k4 as the most similar, sharing
65.9% of copy number segments, followed by kl and k4 at 58.3%, and kl and k2 at 55.0%.
Several cluster-specific CNVs contain genes of potential functional relevance (e). These
include a focal amplification to copy number 6 of IKBKB in cluster kl, which encodes a serine
kinase important in the NF-KB signaling pathway"; another focal amplification to copy number
in cluster kl containing genes DSC1,2,3 and DSG1,2,3,4 all of which encode proteins involved
in cell-cell adhesion and cell positioning and are often mis-regulated in cancer"; and the deletion
of a region containing PDGRFB specific to cluster k2, which encodes a tyrosine kinase cell
surface receptor involved in cell proliferation signaling, and is frequently mutated in cancer'.
Lastly, we applied xSDS SCI-seq to a frozen stage II rectal adenocarcinoma measuring
500 mm3. During preparation we noticed a high abundance of nuclear debris and ruptured nuclei
which likely attributed to the decreased yield of the preparation (16 PCR indexes) of 146 single
cell libraries (median unique read count of 71,378; M50 of 352,168; 111 cells > 50,000 unique
reads). We carried out the same CNV calling approach as with the PDAC sample; however high
frequency breakpoints were not observed and subclonal populations could not be identified (). This may be a result of nuclear deterioration due to irradiation, a common treatment for
rectal cancers, underscoring the challenge of producing high-quality single cell or nuclei
suspensions shared by all single cell methods'.
Discussion
We developed SCI-seq, a method which utilizes nucleosome depletion in a combinatorial
indexing workflow to produce thousands of single cell genome sequencing libraries. Using SCI-
seq, we produced 16,698 single cell libraries (of which 5,395 were sequenced to a depth
sufficient for CNV calling) from myriad samples, including primary tissue isolates representative
of the two major areas of single cell genome research: somatic aneuploidy and cancer. In
addition to the advantages of throughput, the platform does not require specialized microfluidics
equipment or droplet emulsification techniques. Using our more uniform nucleosome depletion
strategy, xSDS, we were able to achieve resolution on the order of 250 kbp, though we suspect
further optimization, such as alternative crosslinking agents, may provide sufficient depth for
improved resolution. We also demonstrate the ability to identify clonal populations that can be
aggregated to facilitate high resolution CNV calling by applying this strategy to a pancreatic
ductal adenocarcinoma which revealed subclone-specific CNVs that may impact proliferation,
migration, or possibly drive other molecular subtypes'.
It may be possible to use this technology to include in situ pre-amplification within the
nuclear scaffold prior to SCI-seq or the incorporation of T4 in vitro transcription, such as in
THS-seq35, an ATAC-seq variant, to boost the resulting coverage and facilitate single nucleotide
variant detection. While optimization is possible, as with any new method, we believe that the
throughput provided by SCI-seq will open the door to deep quantification of mammalian somatic
genome stability as well as serve as a platform to assess other properties of single cells including
DNA methylation and chromatin architecture.
Accession Codes
NCBI BioProject ID: PRJNA326698
He La dbGaP Accession: phs000640
Data Availability
GM12878 and Rhesus sequence data are accessible through the NCBI Sequence Read
Archive (SRA) under BioProject ID: PRJNA326698 for unrestricted access. He La sequence data
are accessible through the database of Genotypes and Phenotypes (dbGaP), as a substudy under
accession number phs000640. Human tumor samples are undergoing submission to dbGaP and
are awaiting study accession assignment. Software developed specifically for this project is
available on the World Wide Web at sci-seq.sourceforge.net.
References cited in Example 1
1. McConnell, M. J. et al. Mosaic Copy Number Variation in Human Neurons. Science (80.).
342, 632-637 (2013).
2. Cai, X. et al. Single-Cell, Genome-wide Sequencing Identifies Clonal Somatic Copy-
Number Variation in the Human Brain. Cell Rep. 8, 1280-1289 (2014).
3. Knouse, K. A., Wu, J., Whittaker, C. A. & Amon, A. Single cell sequencing reveals low
levels of aneuploidy across mammalian tissues. Proc Natl Acad Sci USA 111, 13409-
13414 (2014).
4. Rehen, S. K. et al. Chromosomal variation in neurons of the developing and adult
mammalian nervous system. Proc. Natl. Acad. Sci. U S. A. 98, 13361-6 (2001).
Navin, N. et al. Tumour evolution inferred by single-cell sequencing. Nature 472, 90-94
(2011).
6. Eirew, P. et al. Dynamics of genomic clones in breast cancer patient xenografts at single-
cell resolution. Nature 518, 422-6 (2014).
7. Gawad, C., Koh, W. & Quake, S. R. Dissecting the clonal origins of childhood acute
lymphoblastic leukemia by single-cell genomics. Proc. Natl. Acad. Sci. U S. A. 111,
17947-52 (2014).
8. Gao, R. et al. Punctuated copy number evolution and clonal stasis in triple-negative breast
cancer. Nat. Genet. 1-15 (2016). doi:10.1038/ng.3641
9. Zong, C., Lu, S., Chapman, A. R. & Xie, X. S. Genome-Wide Detection of Single
Nucleotide and Copy Number Variations of a Single Human Cell. Science (80-. ). 338,
1622-1626 (2012).
10. Baslan, T. et al. Optimizing sparse sequencing of single cells for highly multiplex copy
number profiling. Genome Res. 125, 714-724 (2015).
11. Knouse, K. A., Wu, J. & Amon, A. Assessment of megabase-scale somatic copy number
variation using single cell sequencing. Genome Res. gr.198937.115- (2016).
doi:10.1101/gr.198937.115
12. Gawad, C., Koh, W. & Quake, S. R. Single-cell genome sequencing: current state of the
science. Nat. Rev. Genet. 17, 175-88 (2016).
13. Adey, A. et al. Rapid, low-input, low-bias construction of shotgun fragment libraries by
high-density in vitro transposition. Genome Biol. 11, R119 (2010).
14. Amini, S. et al. Haplotype-resolved whole-genome sequencing by contiguity-preserving
transposition and combinatorial indexing. Nat. Genet. 46, 1343-9 (2014).
. Adey, A. et al. In vitro, long-range sequence information for de novo genome assembly
via transposase contiguity. Genome Res. 24, 2041-2049 (2014).
16. Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J.
Transposition of native chromatin for fast and sensitive epigenomic profiling of open
chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213-8
(2013).
17. Cusanovich, D. a et al. Epigenetics. Multiplex single-cell profiling of chromatin
accessibility by combinatorial cellular indexing. Science 348, 910-4 (2015).
18. Stergachis, A. B. et al. Developmental fate and cellular maturity encoded in human
regulatory DNA landscapes. Cell 154, 888-903 (2013).
The ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the
human genome. Nature 489, 57-74 (2012).
. Adey, A. et al. The haplotype-resolved genome and epigenome of the aneuploid HeLa
cancer cell line. Nature 500, 207-211 (2013).
21. Macosko, E. Z. et al. Highly Parallel Genome-wide Expression Profiling of Individual
Cells Using Nano liter Droplets. Cell 161, 1202-1214 (2015).
22. Garvin, T. et al. Interactive analysis and quality assessment of single-cell copy-number
variations. bioRxiv 11346 (2014). doi:10.1101/011346
23. GORYSHIN, I. Y., MILLER, J. A., KIL, Y. V., LANZOV, V. A. & REZNIKOFF, W. S.
Tn5/IS50 target recognition. Proc. Natl. Acad. Sci. USA 95, 10716-10721 (1998).
24. Olshen, A. B., Venkatraman, E. S., Lucito, R. & Wigler, M. Circular binary segmentation
for the analysis of array-based DNA copy number data. Biostatistics 5, 557-572 (2004).
. Ha, G. et al. Integrative analysis of genome-wide loss of heterozygosity and monoallelic
expression at nucleotide resolution reveals disrupted pathways in triple-negative breast
cancer. Genome Res. 22, 1995-2007 (2012).
26. Rosenkrantz, J. & Carbone, L. Investigating somatic aneuploidy in the brain: why we need
a new model. Chromosoma (2016).
27. Callaway, E. 'Platinum' genome takes on disease. Nat. News 515, 323 (2014).
28. Waddell, N. et al. Whole genomes redefine the mutational landscape of pancreatic cancer.
Nature 518, 495-501 (2015).
De Kouchkovsky, I. & Abdul-Hay, M. 'Acute myeloid leukemia: a comprehensive review
and 2016 update'. Blood Cancer 1 6, e441 (2016).
. Kumagai, T. et al. Epigenetic regulation and molecular characterization of C/EBPalpha in
pancreatic cancer cells. Int J Cancer 124, 827-833 (2009).
31. Perkins, N. D. Integrating cell-signalling pathways with NF-kappaB and IKK function.
Nat. Rev. Mol. Cell Biol. 8, 49-62 (2007).
32. Stahley, S. N. & Kowalczyk, A. P. Desmosomes in acquired disease. Cell Tissue Res. 360,
439-56 (2015).
33. Forbes, S. A. et al. COSMIC: Exploring the world's knowledge of somatic mutations in
human cancer. Nucleic Acids Res. 43, D805-D811 (2015).
34. Bailey, P. et al. Genomic analyses identify molecular subtypes of pancreatic cancer.
Nature 531, 47-52 (2016).
. Sos, B. et al. Characterization of chromatin accessibility with a transposome
hypersensitive sites sequencing (THS-seq) assay. Genome Biol 17, 20 (2016).
EXAMPLE 2
Reagents Used in Example 2
Phosphate Buffer Saline (PBS, Thermo Fisher, Cat. 10010023)
0.25% Trypsin (Thermo Fisher, Cat. 15050057)
Tris (Fisher, Cat. T1503)
HC1(Fisher, Cat. A144)
NaC1 (Fisher, Cat. M-11624)
MgC12 (Sigma, Cat. M8226)
Igepal® CA-630 (Sigma, 18896)
Protease Inhibitors (Roche, Cat. 11873580001)
Lithium 3,5-diiodosalicylic acid (Sigma, Cat. D3635) - LAND Only
Formaldehyde (Sigma, Cat. F8775) - xSDS Only
Glycine (Sigma, Cat. G8898) - xSDS Only
HEPES (Fisher, Cat. BP310) - xSDS Only
NEBuffer 2.1 (NEB, Cat. B7202) - xSDS Only
SDS (Sigma, Cat. L3771) - xSDS Only
TritonTM -X100 (Sigma, Cat. 90021) - xSDS Only
DAPI (Thermo Fisher, Cat. D1306)
TD buffer and NPM from Nextera® kit (Illumina, Cat. FC1031)
96 Indexed Transposomes (either assembled using published methods or obtained from
Illumina, oligos shown in Table 4)
Indexed i5 and i7 PCR primers (Table 5)
SYBR Green (FMC BioProducts, Cat. 50513)
Qiaquick® PCR purification kit (Qiagen, Cat. 28104)
dsDNA High Sensitivity qubit (Thermo Fisher, Cat. Q32851)
High Sensitivity Bioanalyzer kit (Agilent, Cat. 5067-4626)
NextSeq sequencing kit (High or Mid 150-cycle)
Sequencing primers (Table 6)
Equipment Used in the Examples
Dounce Homogenizer
35pA4 Cell Strainer (BD Biosciences, Cat. 352235)
Sony SH800 cell sorter (Sony Biotechnology, Cat. SH800) or other FACS instrument
capable of DAPI-based single nuclei sorting
CFX Connect RT Thermal Cycler (Bio-Rad, Cat. 1855200) or other real time
thermocycler
Qubit® 2.0 Flourometer (Thermo Fisher, Cat. Q32866)
2100 Bioanalyzer (Agilent, Cat. G2939A)
NextSeq® 500 (Illumina, Cat. SY1001)
Table 4: Tagmentation Oligos
Name
SEQ ID
Sequence (5'->3')
Mosaic End
/5Phos/CTGTCTCTTATACACATCT
Sequence
TCGTCGGCAGCGTCTCCACGCTATAGCCTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
CPT_TS_i5_1
TCGTCGGCAGCGTCTCCACGCATAGAGGCGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
CPT_TS_i5_2
TCGTCGGCAGCGTCTCCACGCCCTATCCTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
CPT_TS_i5_3
TCGTCGGCAGCGTCTCCACGCGGCTCTGAGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
CPT_TS_i5_4
TCGTCGGCAGCGTCTCCACGCAGGCGAAGGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
CPT_TS_i5_5
TCGTCGGCAGCGTCTCCACGCTAATCTTAGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
CPT_TS_i5_6
TCGTCGGCAGCGTCTCCACGCCAGGACGTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
CPT_TS_i5_7
TCGTCGGCAGCGTCTCCACGCGTACTGACGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
CPT_TS_i5_8
GTCTCGTGGGCTCGGCTGTCCCTGTCCCGAGTAATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_1
GTCTCGTGGGCTCGGCTGTCCCTGTCCTCTCCGGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG 11
CPT_TS_i7_2
GTCTCGTGGGCTCGGCTGTCCCTGTCCAATGAGCGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_3
GTCTCGTGGGCTCGGCTGTCCCTGTCCGGAATCTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_4
GTCTCGTGGGCTCGGCTGTCCCTGTCCTTCTGAATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_5
GTCTCGTGGGCTCGGCTGTCCCTGTCCACGAATTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_6
GTCTCGTGGGCTCGGCTGTCCCTGTCCAGCTTCAGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_7
GTCTCGTGGGCTCGGCTGTCCCTGTCCGCGCATTACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_8
GTCTCGTGGGCTCGGCTGTCCCTGTCCCATAGCCGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_9
GTCTCGTGGGCTCGGCTGTCCCTGTCCTTCGCGGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_10
GTCTCGTGGGCTCGGCTGTCCCTGTCCGCGCGAGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_11
GTCTCGTGGGCTCGGCTGTCCCTGTCCCTATCGCTCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
CPT_TS_i7_12
Table 5: PCR Primers
Name SEQ
Sequence (5'->3') NO
i7-T119-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATaatgccgcttGTCTCGTGGGCTCGG
i7-T120-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATtatagacgcaGTCTCGTGGGCTCGG
i7-T121-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATtcaatcgcatGTCTCGTGGGCTCGG
i7-T122-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATttcttaataaGTCTCGTGGGCTCGG
i7-T123-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATgtcctagaggGTCTCGTGGGCTCGG
i7-T124-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATatattgatacGTCTCGTGGGCTCGG
i7-T125-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATccgctgccagGTCTCGTGGGCTCGG
i7-T126-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATcctagtacgtGTCTCGTGGGCTCGG
i7-T127-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATcaattaccgtGTCTCGTGGGCTCGG
i7-T128-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATggccgtagtcGTCTCGTGGGCTCGG
i7-T129-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATcgattacggcGTCTCGTGGGCTCGG
i7-T130-
N EX2cpt-
A CAAGCAGAAGACGGCATACGAGATtaatgaacgaGTCTCGTGGGCTCGG
i7-T131-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATccgttccttaGTCTCGTGGGCTCGG
i7-T132-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATggtaccatatGTCTCGTGGGCTCGG
i7-T133-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATccgattcgcaGTCTCGTGGGCTCGG
i7-T134-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATatggctctgcGTCTCGTGGGCTCGG
i7-T135-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATgtataatacgGTCTCGTGGGCTCGG
i7-T136-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATatcagcaagtGTCTCGTGGGCTCGG
i7-T137-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATggcgaactcgGTCTCGTGGGCTCGG
i7-T138-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATttaattgaatGTCTCGTGGGCTCGG
i7-T139-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATttaggaccggGTCTCGTGGGCTCGG
i7-T140-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATaagtaagagcGTCTCGTGGGCTCGG
i7-T141-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATccttggtccaGTCTCGTGGGCTCGG
i7-T142-
N EX2cpt-
B CAAGCAGAAGACGGCATACGAGATcatcagaatgGTCTCGTGGGCTCGG
i7-T143-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATttatagcagaGTCTCGTGGGCTCGG
i7-T144-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATttacttggaaGTCTCGTGGGCTCGG
i7-T145-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATgctcagccggGTCTCGTGGGCTCGG
i7-T146-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATacgtccgcagGTCTCGTGGGCTCGG
i7-T147-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATttgactgacgGTCTCGTGGGCTCGG
i7-T148-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATttgcgaggcaGTCTCGTGGGCTCGG
i7-T149-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATttccaaccgcGTCTCGTGGGCTCGG
i7-T150-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATtaaccttcggGTCTCGTGGGCTCGG
i7-T151-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATtcaagccgatGTCTCGTGGGCTCGG
i7-T152-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATcttgcaacctGTCTCGTGGGCTCGG
i7-T153-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATccatcgcgaaGTCTCGTGGGCTCGG
i7-T154-
N EX2cpt-
C CAAGCAGAAGACGGCATACGAGATtagacttcttGTCTCGTGGGCTCGG
i7-T231-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATtgcgcgatgcGTCTCGTGGGCTCGG
i7-T232-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATattgagattgGTCTCGTGGGCTCGG
i7-T233-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATttgatatattGTCTCGTGGGCTCGG
i7-T234-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATcggtaggaatGTCTCGTGGGCTCGG
i7-T235-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATaccagcgcagGTCTCGTGGGCTCGG
i7-T236-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATcgaatgagctGTCTCGTGGGCTCGG
i7-T237-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATagttcgagtaGTCTCGTGGGCTCGG
i7-T238-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATttggacgctgGTCTCGTGGGCTCGG
i7-T239-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATatagactaggGTCTCGTGGGCTCGG
i7-T240-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATtatagtaagcGTCTCGTGGGCTCGG
i7-T241-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATcggtcgttaaGTCTCGTGGGCTCGG
i7-T242-
N EX2cpt-
D CAAGCAGAAGACGGCATACGAGATatggcggatcGTCTCGTGGGCTCGG
i7-T243-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATctctgatcagGTCTCGTGGGCTCGG
i7-T244-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATggccagtccgGTCTCGTGGGCTCGG
i7-T245-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATcggaagatatGTCTCGTGGGCTCGG
i7-T246-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATtggctgatgaGTCTCGTGGGCTCGG
i7-T247-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATgaaggttgccGTCTCGTGGGCTCGG
i7-T248-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATgttgaaggatGTCTCGTGGGCTCGG
i7-T249-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATccattcgtaaGTCTCGTGGGCTCGG
i7-T250-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATtgcgccagaaGTCTCGTGGGCTCGG
i7-T251-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATcgaataattcGTCTCGTGGGCTCGG
i7-T252-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATgcgacgccttGTCTCGTGGGCTCGG
i7-T253-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATatcaacgattGTCTCGTGGGCTCGG
i7-T254-
N EX2cpt-
E CAAGCAGAAGACGGCATACGAGATgttctgaattGTCTCGTGGGCTCGG
i7-T255-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATgctaacctcaGTCTCGTGGGCTCGG
i7-T256-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATcaagcaactgGTCTCGTGGGCTCGG
i7-T257-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATggagcggccgGTCTCGTGGGCTCGG
i7-T258-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATcgcgtacgacGTCTCGTGGGCTCGG
i7-T259-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATcgatggcgccGTCTCGTGGGCTCGG
i7-T260-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATtggtattcatGTCTCGTGGGCTCGG
i7-T261-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATgataaggcaaGTCTCGTGGGCTCGG
i7-T262-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATgccggtcgagGTCTCGTGGGCTCGG
i7-T263-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATtgcgccatctGTCTCGTGGGCTCGG
i7-T264-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATaagtcttccgGTCTCGTGGGCTCGG
i7-T265-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATagactcaagcGTCTCGTGGGCTCGG
i7-T266-
N EX2cpt-
F CAAGCAGAAGACGGCATACGAGATgcaggcgacgGTCTCGTGGGCTCGG
i5-T155-
NEX1cpt-
A AATGATACGGCGACCACCGAGATCTACACgtccttaagaTCGTCGGCAGCGTC
i5-T156-
NEX1cpt-
A AATGATACGGCGACCACCGAGATCTACACagtaacggtcTCGTCGGCAGCGTC
i5-T157-
NEX1cpt-
A AATGATACGGCGACCACCGAGATCTACACgttcgtcagaTCGTCGGCAGCGTC
i5-T158-
NEX1cpt-
A AATGATACGGCGACCACCGAGATCTACACcgcctaatgcTCGTCGGCAGCGTC
i5-T159-
NEX1cpt-
A AATGATACGGCGACCACCGAGATCTACACaccggaattaTCGTCGGCAGCGTC
i5-T160-
NEX1cpt-
A AATGATACGGCGACCACCGAGATCTACACtaggccatagTCGTCGGCAGCGTC
i5-T161-
NEX1cpt-
A AATGATACGGCGACCACCGAGATCTACACtaactcttagTCGTCGGCAGCGTC
i5-T162-
NEX1cpt-
A AATGATACGGCGACCACCGAGATCTACACtatgagttaaTCGTCGGCAGCGTC
i5-T163-
NEX1cpt-
B AATGATACGGCGACCACCGAGATCTACACtatcatgatcTCGTCGGCAGCGTC
i5-T164-
NEX1cpt-
B AATGATACGGCGACCACCGAGATCTACACgagcatatggTCGTCGGCAGCGTC
i5-T165-
NEX1cpt-
B AATGATACGGCGACCACCGAGATCTACACtaacgatccaTCGTCGGCAGCGTC
i5-T166-
NEX1cpt-
B AATGATACGGCGACCACCGAGATCTACACcggcgtaactTCGTCGGCAGCGTC
i5-T167-
NEX1cpt-
B AATGATACGGCGACCACCGAGATCTACACcgtcgcagccTCGTCGGCAGCGTC
i5-T168-
NEX1cpt-
B AATGATACGGCGACCACCGAGATCTACACgtagctccatTCGTCGGCAGCGTC
i5-T169-
NEX1cpt-
B AATGATACGGCGACCACCGAGATCTACACttgccttggcTCGTCGGCAGCGTC
i5-T170-
NEX1cpt-
B AATGATACGGCGACCACCGAGATCTACACtgctaattctTCGTCGGCAGCGTC
i5-T171-
NEX1cpt-
C AATGATACGGCGACCACCGAGATCTACACgtcctacttgTCGTCGGCAGCGTC
i5-T172-
NEX1cpt-
C AATGATACGGCGACCACCGAGATCTACACggtaggttagTCGTCGGCAGCGTC
i5-T173-
NEX1cpt-
C AATGATACGGCGACCACCGAGATCTACACgagcatcattTCGTCGGCAGCGTC
i5-T174-
NEX1cpt-
C AATGATACGGCGACCACCGAGATCTACACccgctccggcTCGTCGGCAGCGTC
i5-T175-
NEX1cpt-
C AATGATACGGCGACCACCGAGATCTACACttcttccggtTCGTCGGCAGCGTC
i5-T176-
NEX1cpt-
C AATGATACGGCGACCACCGAGATCTACACaggagagaacTCGTCGGCAGCGTC
i5-T177-
NEX1cpt-
C AATGATACGGCGACCACCGAGATCTACACtaactcaattTCGTCGGCAGCGTC
i5-T178-
NEX1cpt-
C AATGATACGGCGACCACCGAGATCTACACactataggttTCGTCGGCAGCGTC
i5-T207-
NEX1cpt-
D AATGATACGGCGACCACCGAGATCTACACtaacgaattgTCGTCGGCAGCGTC
i5-T208-
NEX1cpt-
D AATGATACGGCGACCACCGAGATCTACACtgagaaccaaTCGTCGGCAGCGTC
i5-T209-
NEX1cpt-
D AATGATACGGCGACCACCGAGATCTACACttattctgagTCGTCGGCAGCGTC
i5-T210-
NEX1cpt-
D AATGATACGGCGACCACCGAGATCTACACttattatggtTCGTCGGCAGCGTC
i5-T211-
NEX1cpt-
D AATGATACGGCGACCACCGAGATCTACACatatgagccaTCGTCGGCAGCGTC
i5-T212-
NEX1cpt-
D AATGATACGGCGACCACCGAGATCTACACcaaccagtacTCGTCGGCAGCGTC
i5-T213-
NEX1cpt-
D AATGATACGGCGACCACCGAGATCTACACcatccgactaTCGTCGGCAGCGTC
i5-T214-
NEX1cpt-
D AATGATACGGCGACCACCGAGATCTACACatcatggctgTCGTCGGCAGCGTC
i5-T215-
NEX1cpt-
E AATGATACGGCGACCACCGAGATCTACACccgcaagttcTCGTCGGCAGCGTC
i5-T216-
NEX1cpt-
E AATGATACGGCGACCACCGAGATCTACACcttctcattgTCGTCGGCAGCGTC
i5-T217-
NEX1cpt-
E AATGATACGGCGACCACCGAGATCTACACcaggaggagaTCGTCGGCAGCGTC
i5-T218-
NEX1cpt-
E AATGATACGGCGACCACCGAGATCTACACgatatcggcgTCGTCGGCAGCGTC
i5-T219-
NEX1cpt-
E AATGATACGGCGACCACCGAGATCTACACccagtcctctTCGTCGGCAGCGTC
i5-T220-
NEX1cpt-
E AATGATACGGCGACCACCGAGATCTACACcatagttcggTCGTCGGCAGCGTC
i5-T221-
NEX1cpt-
E AATGATACGGCGACCACCGAGATCTACACcgtaatgcagTCGTCGGCAGCGTC
i5-T222-
NEX1cpt-
E AATGATACGGCGACCACCGAGATCTACACccgttcggatTCGTCGGCAGCGTC
i5-T223-
NEX1cpt-
F AATGATACGGCGACCACCGAGATCTACACccataagtccTCGTCGGCAGCGTC
i5-T224-
NEX1cpt-
F AATGATACGGCGACCACCGAGATCTACACggcaatgagaTCGTCGGCAGCGTC
i5-T225-
NEX1cpt-
F AATGATACGGCGACCACCGAGATCTACACcggttatgccTCGTCGGCAGCGTC
i5-T226-
NEX1cpt-
F AATGATACGGCGACCACCGAGATCTACACtggccggcctTCGTCGGCAGCGTC
i5-T227-
NEX1cpt-
F AATGATACGGCGACCACCGAGATCTACACagctgcaataTCGTCGGCAGCGTC
i5-T228-
NEX1cpt-
F AATGATACGGCGACCACCGAGATCTACACtggccatgcaTCGTCGGCAGCGTC
i5-T229-
NEX1cpt-
F AATGATACGGCGACCACCGAGATCTACACtgacgctccgTCGTCGGCAGCGTC
i5-T230-
NEX1cpt-
F AATGATACGGCGACCACCGAGATCTACACaactgctgccTCGTCGGCAGCGTC
Table 6: Seauencina Primers
Name Sequence (5'->3') SEQ ID NO
GCGATCGAGGACGGCAGATGTGTATAAGAGACAG 142
Read 1 sequencing primer
Read 2 sequencing primer CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
Index 1 sequencing primer CTGTCTCTTATACACATCTGAGGCGGAGACGGTG
Index 2 sequencing primer CTGTCTCTTATACACATCTGCCGTCCTCGATCGC
I. Preparation of Nuclei Using Lithium 3,5-diiodosalicylic acid (LAND) or SDS (xSDS)
A. LAND Method of Nuclei Preparation & Nucleosome Depletion
If the cells were in a suspension cell culture, the culture was gently triturated to break up
cell clumps, the cells were pelleted by spinning at 500xg for 5 minutes at 4°C, and washed with
500 !IL ice cold PBS.
If the cells were in an adherent cell culture, media was aspirated and the cells washed
with 10 nth of PBS at 37°C, and then enough 0.25% Trypsin at 37°C was added to cover the
monolayer. After incubating at 37°C for 5 minutes or until 90% of cells were no longer adhering
to the surface, 37°C media was added at 1:1 ratio to quench Trypsin. The cells were pelleted by
spinning at 500xg for 5 minutes at 4°C, and then washed with 500 [iL ice cold PBS.
If a tissue was used, the tissue sample was placed in a 2 mL dounce homoginzer on ice.
Two mls of NIB buffer (10mM TrisHC1 pH7.4, 10MM NaCl, 3mM MgCl2, 0.1% Igepal®, lx
protease inhibitors) were added to the sample and incubated on ice for 5 minutes. The sample
was dounced 5 times with loose pestle followed by 15 strokes with tight pestle, and then put
through a 351.tM cell strainer, and additional strainers were used as necessary.
The cells from either suspension cell culture, adherent cell culture, or tissue sample were
pelleted by spinning at 500xg for 5 minutes, and then resuspended in 200 tiL 12.5 mM LIS in
NIB buffer (2.5 UL IM US + 197.5 !IL NIB buffer). After incubating on ice for 5 minutes, 800
[IL NIB buffer and 5 pL DAPI (5 mg/mL) were added. The cells were gently passed through a
351tM cell strainer.
B. xSDS Method of Nuclei Preparation & Nucleosome Depletion
If the cells were in a suspension cell culture, the medium was gently triturated to break up
cell clumps. To 10 mL of cells in media 406 ith of 37% formaldehyde were added and incubated
at room temp for 10 minutes with gentle shaking. Eight hundred microliters of 2.5 M Glycine
were added to the cells and incubated on ice for 5 minutes, and then centrifuged at 550xg for 8
minutes at 4°C. After washing with 10 nth of ice cold PBS, the cells were resuspended in 5 nth
of ice cold NIB (10mM TrisHCA p117.4, 10mM NaC1, 3mkt MgC12, 0.1% Igepalt, lx protease
inhibitors), and incubated on ice for 20 minutes with gentle mixing.
If the cells were in an adherent cell culture, media was aspirated and the cells washed
with 10 mL of PBS at 37°C, and then enough 0.25% Trypsin at 37°C was added to cover the
monolayer. After incubating at 37°C for 5 minutes or until 90% of cells were no longer adhering
to the surface, 37°C media was added at 1:1 ratio to quench Trypsin, and the volume brought to
10m1 with media. The cells were resuspended in 10 mL media, and 406 [IL of 37%
formaldehyde added and incubated at room temp for 10 minutes with gentle shaking. Eight
hundred microliters of 2.5 M Glycine were added to the cells and incubated on ice for 5 minutes.
The cells were centrifuged at 550xg, for 8 minutes at 4° and washed with 10 mL of ice cold PBS.
After resuspending the cells in 5 mL of ice cold NIB, they were incubated on ice for 20 minutes
with gentle mixing.
If a tissue was used, the tissue sample was placed in a 2 mL Dounce homogenizer on ice.
Two rnLs of HEPES NIB (20mM HEPES, 10MNI NaC1, 3mM MgC1.2, 0.1% igepal, 1.x protease
inhibitors) buffer were added to the sample and incubated on ice for 5 minutes. The sample was
dounced 5 times with loose pestle followed by 15 strokes with tight pestle, and then put through
a 35pA4 cell strainer, and additional strainers were used as necessary. The volume was brought
up to 10m1 with HEPES-NIB, and 406 4, of 37% formaldehyde were added to the 10 mL
volume. Eight hundred microliters of 2.5 M Glycine were added and incubated on ice 5 minutes.
The cells or nuclei from either suspension cell culture or adherent cell culture were
pelleted by spinning at 500xg for 5 minutes and washed with 900 pL of lx NEBuffer 2.1. After
spinning at 500 x g for 5 minutes, the pellet was resuspended in 800 lmL lx NEBuffer 2.1 with 12
UL of 20% SDS and incubated at 42°C with vigorous shaking for 30 minutes, and then 200 pl. of
% TritonTm X-100 was added and incubated at 42°C with vigorous shaking for 30 minutes.
The cells were gently passed through a 3511M cell strainer, and 5 pL DAN (5 mg/mL) was
added.
II, Nuclei Sorting and Tagmentati on
A tagmentation plate was prepared with 10 [IL ix TD buffer (for 1 plate: 500 pt NIB
buffer + 500 [IL TD buffer), and 2000 single nuclei were sorted into each well of the
tagmentation plate. At this step the number of nuclei per well can be varied slightly as long as
the number of nuclei per well is consistent for the whole plate. It is also possible to multiplex
different samples into different wells of the plate as the transposase index will be preserved. The
cells were gated according to Figure 33, After spinning down the plate, 1 1tL 2.5 nM of uniquely
indexed transposome were added to each well. After sealing, the plate was incubated at 55°C for
minutes with gentle shaking. The plate was then returned to room temperature and then
placed on ice. All the wells were pooled, 5 p1 DAPI (5 mg/mL) were added and then the cells
were passed through a 3504 cell strainer.
Second Sort of and PCR Indexing
A master mix was prepared for each well with 0.25 [IL 20mg/mL BSA, 0.5 pt 1% SDS,
and 7.75 IAL H20. Master mix (8.5 !_t1_,) and 2.5 !IL of each (i5 and i7) 10 [1.1\4 primer was added
to each well of a 96 well plate. Single nuclei (15-22) were sorted into each well using the most
stringent sort settings. The plate was then spun down. Those nuclei prepared using the LAND
method were incubated for 5 minutes at 55° to denature transposase. Those nuclei prepared
using the xSDS method were incubated at 68° for 45 minutes to denature transposase and reverse
crosslinks.
Buffer was prepared (for 1 plate: 7500, -NPM, 400 Id, H20, and 50 !IL 100x SYBR
Green), and 12pL. of the buffer was added to each well of strip tube. The following PCR cycles
were performed: 72°C for 5 minutes, 98°C for 30 seconds, then continual cycles of (98°C for 10
seconds, 63°C for 30 seconds, 72°C for one minute followed by a plate read and an additional 10
seconds at 72°C). These cycles were repeated until the majority of wells exhibited exponential
amplification as determined by SYBR green fluorescence.
IV. Library Clean Up and Quantification
Libraries were pooled using 5 uL of each well of the PCR plate, then purified using a
Qiaquick® PCR Purification column and eluted in 30 pL of 10 mM Tris-Cl, pH 8.5 (EB). Two
microliters were used to quantify the concentration of DNA with dsDNA High Sensitivity
Qubit® 2.0 Fluorometer, following the manufacturer's protocol. The Qubit® readout was used
to dilute library to -4 ng/uL, and 1 uL was run on a High Sensitivity Bioanalyser 2100,
following the manufacturer's protocol. The library was then quantified for the 200bp - 1 kbp
range to dilute the pool to 1 nM for Illumina Sequencing.
V. Sequencing
A NextSeq® 500 was set up for a run as per manufacturer's instructions for a 1 nM
sample except for the following changes. The library pool was loaded at a concentration of 0.8
pM and a total volume of 1.5 mL and deposited into cartridge position 10; custom primers were
setup by diluting 9 uL of 100 iM stock sequencing primer 1 into a total of 1.5 mL of HT 1 buffer
into cartridge position 7; sequencing primer was setup by diluting 9 p.1_, of 100 uM stock
sequencing primer 2 into a total of 1.5 mL of HT1 buffer into cartridge position 8; and custom
index sequencing primers were setup by diluting 18 tL of each custom index sequencing primer
at 100 pM stock concentrations into a total of 3 mL of HT1 buffer into cartridge position 9 (see
Table 7). The NextSeq® 500 was operated in standalone mode; the SCIseq custom chemistry
recipe (Amini et al., 2014, Nat. Genet,
46. 1343---1349) was selected; dual index was selected; the
appropriate number of read cycles was entered (50 recommended) and 18 cycles for each index;
the custom checkbox for all reads and indices was selected.
Table 7:
Cartridge Reagent Concentration Total Volume Stock oligo HT1
position (dilute in HT1) (100 uM)
7 Custom Read 1 0.6 uM 1.5 mL 9 )1,1_, 1491
8 Custom Read 2 0.6 uM 1.5 mL 9 )1,1_, 1491
Custom Index 1 & 2 Each 0.6 uM 3 mL 18 [EL 2964
each
Library 0.8 pM (<800 bp) 1.5 mL
EXAMPLE 3
Single-cell combinatorial indexing and genome and chromosome conformation
Restriction endonuclease digestion of isolated nuclei followed by ligation can be used to
acquire information on chromosome structure within a nucleus, such as chromatin folding
analysis and detection of genomic rearrangements. Such types of analyses are known in art as
chromosome conformation capture (3C) and related methods (4C, 5C, and Hi-C).
The method of single-cell combinatorial indexing and genome and chromosome
conformation (sci-GCC) that can be used in conjunction with the method described in Examples
1 and 2 is described in Figure 34. Specifically, the method of single-cell combinatorial indexing
and genome and chromosome conformation includes blocks 12, 13, 14, and 19 as shown in
Figure 34. Unlike other methods of genome and chromosome conformation analysis of single
cells (Nagano et al., 2013, Nature, 502:59-64), the method described herein does not require
biotin fill-in or biotin pull-down so as to obtain both genome and chromatin conformation
sequence data.
Conditions for cross-linking cells were evaluated to determine the minimum
concentration of formaldehyde needed to cross-link cells and maintain nuclei integrity. HeLa
cells were cross-linked by exposing the cells to formaldehyde at 0.2%, 0.35%, 1.5%, or no
formaldehyde, and an abbreviated version of the method described in Figure 34 was done and the
number of nuclei resulting was determined.
No intact nuclei were isolated from cells not exposed to formaldehyde or exposed to
0.2% formaldehyde. Cells exposed to 0.35% formaldehyde yielded 3.8 x 105 nuclei with normal
morphology, and cells exposed to 1.5% formaldehyde yielded 6.4 x 105 nuclei with normal
morphology.
Conditions for reversing cross-linking was also evaluated. HeLa cells were cross-linked
by exposing the cells to formaldehyde at 0.35%, 0.75%, 1.5%, or no formaldehyde, and an
abbreviated version of the method described in Figure 34 was performed. Cross-linking was
revered by incubating isolated nuclei at 68°C for with 1 hour or 16 hours (Figure 35).
The data indicate that the use of 0.35% formaldehyde with reversal conditions of 1 hour
incubation at 68°C was best.
From sequenced sci-GCC libraries comparable unique read counts genome wide were
obtained as in methods described in Examples 1 and 2 and Figure 35. In addition to the genomic
sequence reads, between 5% and 15% of sequence reads contained chimeric ligation junctions
that were characteristic of chromatin conformation signal as described in Nagano et al., (2013,
Nature, 502:59-64). On average, we obtained an increased unique chimeric ligation junction read
count when compared with existing single cell HiC strategies (see, for instance, Nagano et al.,
2013, Nature, 502:59-64) with a mean unique chimeric ligation junction read count of over
40,000 per cell in crosslinking-optimized preparations. On HeLa, these libraries produced
sufficient chimeric ligation junction reads to clearly identify chromatin structure, including a
known translocation in HeLa (Figure 36).
The complete disclosure of all patents, patent applications, and publications, and
electronically available material (including, for instance, nucleotide sequence submissions in,
e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF,
PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are
incorporated by reference in their entirety. Supplementary materials referenced in publications
(such as supplementary tables, supplementary figures, supplementary materials and methods,
and/or supplementary experimental data) are likewise incorporated by reference in their entirety.
In the event that any inconsistency exists between the disclosure of the present application and
the disclosure(s) of any document incorporated herein by reference, the disclosure of the present
application shall govern. The foregoing detailed description and examples have been given for
clarity of understanding only. No unnecessary limitations are to be understood therefrom. The
invention is not limited to the exact details shown and described, for variations obvious to one
skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular
weights, and so forth used in the specification and claims are to be understood as being modified
in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the
numerical parameters set forth in the specification and claims are approximations that may vary
depending upon the desired properties sought to be obtained by the present invention. At the
very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims,
each numerical parameter should at least be construed in light of the number of reported
significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope
of the invention are approximations, the numerical values set forth in the specific examples are
reported as precisely as possible. All numerical values, however, inherently contain a range
necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the
meaning of the text that follows the heading, unless so specified.
Claims (50)
- What is claimed is: A method of preparing a sequencing library comprising nucleic acids from a plurality of single cells, the method comprising: (a) providing isolated nuclei from a plurality of cells; (b) subjecting the isolated nuclei to a chemical treatment to generate nucleosome- depleted nuclei, while maintaining integrity of the isolated nuclei; (c) distributing subsets of the nucleosome-depleted nuclei into a first plurality of compartments and contacting each subset with a transposome complex, wherein the transposome complex in each compartment comprises a transposase and a first index sequence that is different from first index sequences in the other compartments; (d) fragmenting nucleic acids in the subsets of nucleosome-depleted nuclei into a plurality of nucleic acid fragments and incorporating the first index sequences into at least one strand of the nucleic acid fragments to generate indexed nuclei comprising indexed nucleic acid fragments, wherein the indexed nucleic acid fragments remain attached to the transposases; (e) combining the indexed nuclei to generate pooled indexed nuclei; distributing subsets of the pooled indexed nuclei into a second plurality of compartments; incorporating into the indexed nucleic acid fragments in each compartment a second index sequence to generate dual-index fragments, wherein the second index sequence in each compartment is different from second index sequences in the other compartments; (h) combining the dual-index fragments, thereby producing a sequencing library comprising whole genome nucleic acids from the plurality of single cells.
- 2. The method of claim 1, wherein the chemical treatment comprises a treatment with a chaotropic agent capable of disrupting nucleic acid-protein interactions.
- The method of claim 2, wherein the chaotropic agent comprises lithium 3,5- diiodosalicylic acid.
- 4. The method of claim 1, wherein the chemical treatment comprises a treatment with a detergent capable of disrupting nucleic acid-protein interactions.
- 5. The method of claim 4, wherein the detergent comprises sodium dodecyl sulfate (SDS).
- 6. The method of claim 5, wherein the nuclei are treated with the cross-linking agent prior to step (b).
- 7. The method of claim 6, wherein the cross-linking agent is formaldehyde.
- 8. The method of claim 7, wherein the concentration of formaldehyde ranges from about 0.2% to about 2%.
- 9. The method of claim 7, wherein the concentration of formaldehyde is no greater than about 1.5%.
- 10. The method of claim 7, wherein the cross-linking by formaldehyde is reversed after step (f) and prior to step (g).
- 11. The method of claim 10, wherein the reversal of the cross-linking comprises incubation at about 55°C to about 72°C.
- 12. The method of any one of claims 10 or 11, wherein the transposases are disassociated from the indexed nucleic acid fragments prior to the reversal of the cross-linking.
- 13. The method of claim 12, wherein the transposases are disassociated from the indexed nucleic acid fragments using sodium dodecyl sulfate (SDS).
- 14. The method of claim 1, wherein the nuclei are treated with a restriction enzyme prior to step (d).
- 15. The method of claim 14, wherein the nuclei are treated with a ligase after treatment with the restriction enzyme.
- 16. The method of claim 1, wherein the distributing in steps (c) and (f) is performed by fluorescence-activated nuclei sorting.
- The method of claim 1, wherein the subsets of the nucleosome-depleted nuclei comprise approximately equal numbers of nuclei.
- 18. The method of claim 17, wherein the subsets of the nucleosome-depleted nuclei comprise from 1 to about 2000 nuclei.
- 19. The method of claim 1, wherein the first plurality of compartments is a multi-well plate.
- 20. The method of claim 19, wherein the multi-well plate is a 96-well plate or a 384-well plate.
- The method of claim 1, wherein the subsets of the pooled indexed nuclei comprise approximately equal numbers of nuclei.
- 22. The method of claim 21, wherein the subsets of the pooled indexed nuclei comprise from 1 to about 25 nuclei.
- 23. The method of claim 1, wherein the subsets of the pooled indexed nuclei include at least 10 times fewer nuclei than the subsets of the nucleosome-depleted nuclei.
- 24. The method of claim 1, wherein the subsets of the pooled indexed nuclei include at least 100 times fewer nuclei than the subsets of the nucleosome-depleted nuclei.
- 25. The method of claim 1, wherein the second plurality of compartments is a multi-well plate.
- 26. The method of claim 25, wherein the multi-well plate is a 96-well plate or a 384-well plate.
- 27. The method of claim 1, wherein step (c) comprises adding the transposome complex to the compartments after the subsets of nucleosome-depleted nuclei are distributed.
- 28. The method of claim 1, wherein each of the transposome complexes comprises a transposon, each of the transposons comprising a transferred strand.
- 29. The method of claim 28, wherein the transferred strand comprises the first index sequence and a first universal sequence.
- 30. The method of claim 29, wherein the incorporation of the second index sequence in step (g) comprises contacting the indexed nucleic acid fragments in each compartment with a first universal primer and a second universal primer, each comprising an index sequence and each comprising a sequence identical to or complementary to a portion of the first universal sequence, and performing an exponential amplification reaction.
- 31. The method of claim 30, wherein the index sequence of the first universal primer is the reverse complement of the index sequence of the second universal primer.
- 32. The method of claim 30, wherein the index sequence of the first universal primer is different from the reverse complement of the index sequence of the second universal primer.
- 33. The method of claim 30, wherein the first universal primer further comprises a first capture sequence and a first anchor sequence complementary to a universal sequence at the 3' end of the dual-index fragments.
- 34. The method of any one of claims 30 to 33, wherein the first capture sequence comprises the P5 primer sequence.
- 35. The method of claim 30, wherein the second universal primer further comprises a second capture sequence and a second anchor sequence complementary to a universal sequence at the 5' end of the dual-index fragments.
- 36. The method of claim 35, wherein the second capture sequence comprises the reverse complement of the P7 primer sequence.
- 37. The method of claim 30, wherein the exponential amplification reaction comprises a polymerase chain reaction (PCR).
- 38. The method of claim 37, wherein the PCR comprises 15 to 30 cycles.
- 39. The method of claim 1, further comprising an enrichment of dual-index fragments using a plurality of capture oligonucleotides having specificity for the dual-index fragments.
- 40. The method of claim 39, wherein the capture oligonucleotides are immobilized on a surface of a solid substrate.
- The method of any one of claims 39 to 40, wherein the capture oligonucleotides comprise a first member of a universal binding pair, and wherein a second member of the binding pair is immobilized on a surface of a solid substrate.
- 42. The method of claim 1, further comprising sequencing of the dual-index fragments to determine the nucleotide sequence of nucleic acids from the plurality of single cells.
- 43. The method of claim 42, further comprising: providing a surface comprising a plurality of amplification sites, wherein the amplification sites comprise at least two populations of attached single stranded capture oligonucleotides having a free 3' end, and contacting the surface comprising amplification sites with the dual-index fragments under conditions suitable to produce a plurality of amplification sites that each comprise a clonal population of amplicons from an individual dual-index fragment.
- 44. The method of claim 43, wherein the number of the dual-index fragments exceeds the number of amplification sites, wherein the dual-index fragments have fluidic access to the amplification sites, and wherein each of the amplification sites comprises a capacity for several dual-index fragments in the sequencing library.
- 45. The method of any one of claims 43 to 44, wherein the contacting comprises simultaneously (i) transporting the dual-index fragments to the amplification sites at an average transport rate, and (ii) amplifying the dual-index fragments that are at the amplification sites at an average amplification rate, wherein the average amplification rate exceeds the average transport rate.
- 46. A composition comprising chemically treated nucleosome-depleted isolated nuclei, wherein the isolated nuclei comprise indexed nucleic acid fragments.
- 47. The composition of claim 46, wherein the isolated nuclei comprise non-natural cross- links.
- 48. The composition of claim 46, wherein the composition comprises indexed nucleic acid fragments that terminate in a cleaved restriction site comprising an overhang.
- 49. The composition of any one of claims 46 to 48, wherein the isolated nuclei comprise rearranged genomic DNA.
- 50. A multi-well plate, wherein a well of the multi-well plate comprises the composition of any one of claims 46-49. 1172 Provide isolated nuclei Deplete nucleosomes Distribute subsets. o nucleosome- depleted nuclei Index nuclei by tagmentation 16 Pool and distribute indexed nuclei index fragments by PCR or adapter igation 18 Immobilize library and sequence 3172 371-01. 4.A .17.07. 4B Tissue Brain Tumor tissue tissue culture IU:k et 8 v81N Combine Index 1: transposase based Sort 15-22 per well Index 2: PCR based ChrI 6...2.006k415-Z.041 r. ' :.: - q22 I q223 q23.2 q24 1q2 p12.3 p12,1 pill 01,1 43171 q12 I c1122 q21 tandard 7,°] LAND xSDS . ;41 A)*****146Aei404.)409066,4 l'ieA,4,)§0445P44.:X 41104t94,1000LA*44440,4,4,A44040404.444444,04,00 444 - :MN* SYNG NOX01 en es GFER TBL3 NDUF8IO RPS2 SNI-IGg RNIF161 FNCO.P:F p0,1:0 JI 4C c809< cD 0, ' ..,-- . 800-- )4663 5 518-, 600- 373-- a3 400- 200- 600 800 200 400 165 0 x1,000 DAPI-A-Compensated FSC-A 3.0- 162 104 106 c305. x256 600 208 5 , ,;44 ,- ;e Y`i"g c.0) cn - ea 159., :g co 400 1 1 - 200- yiiy 200 400 i)tio 10 DAP H-Compensated FSC-A 9157. 732 , 549 - F16'. 5B 1'04 105 106 DAP -A-Compensated
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US201662365916P | 2016-07-22 | 2016-07-22 | |
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US62/451,305 | 2017-01-27 | ||
PCT/US2017/043381 WO2018018008A1 (en) | 2016-07-22 | 2017-07-21 | Single cell whole genome libraries and combinatorial indexing methods of making thereof |
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