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Integrated genome and transcriptome sequencing of the same cell.

Dey SS, Kester L, Spanjaard B, Bienko M, van Oudenaarden A - Nat. Biotechnol. (2015)

Bottom Line: We describe a quasilinear amplification strategy to quantify genomic DNA and mRNA from the same cell without physically separating the nucleic acids before amplification.We show that the efficiency of our integrated approach is similar to existing methods for single-cell sequencing of either genomic DNA or mRNA.Further, we find that genes with high cell-to-cell variability in transcript numbers generally have lower genomic copy numbers, and vice versa, suggesting that copy number variations may drive variability in gene expression among individual cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences), Utrecht, the Netherlands. [2] University Medical Center Utrecht, Cancer Genomics Netherlands, Utrecht, the Netherlands.

ABSTRACT
Single-cell genomics and single-cell transcriptomics have emerged as powerful tools to study the biology of single cells at a genome-wide scale. However, a major challenge is to sequence both genomic DNA and mRNA from the same cell, which would allow direct comparison of genomic variation and transcriptome heterogeneity. We describe a quasilinear amplification strategy to quantify genomic DNA and mRNA from the same cell without physically separating the nucleic acids before amplification. We show that the efficiency of our integrated approach is similar to existing methods for single-cell sequencing of either genomic DNA or mRNA. Further, we find that genes with high cell-to-cell variability in transcript numbers generally have lower genomic copy numbers, and vice versa, suggesting that copy number variations may drive variability in gene expression among individual cells. Applications of our integrated sequencing approach could range from gaining insights into cancer evolution and heterogeneity to understanding the transcriptional consequences of copy number variations in healthy and diseased tissues.

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Related in: MedlinePlus

Schematic of DR-Seq for sequencing gDNA and mRNA from the same cell. (a) gDNA and mRNA/cDNA are shown in red and green, respectively. Following single-cell lysis and RT using adapter Ad-1x (purple), gDNA and single stranded cDNA are amplified by Ad-2 (blue) using a quasilinear amplification strategy. The majority of the short amplicons contain Ad-2 at both ends and cDNA-derived amplicons contain Ad-2 at one end and Ad-1x at the other end. The sample is then split into two halves and processed separately to amplify and sequence gDNA or cDNA. (b) Distribution of reads within 100 nucleotides of the gene Dppa5a for two single cells (red and black) as a function of the random priming location by adapter Ad-2. The unique length-based identifiers found in the two cells can be used to count the original number of cDNA molecules within each cell and minimize amplification biases. The figure shows that distinct positions are randomly primed within each cell with high affinity binding sites being preferentially primed. The size of the dots indicate the binding propensity of each location. For most genes such as Dppa5a, the number of theoretical binding sites far exceed the number of length-based identifiers detected, thereby enabling length-based identifiers to accurately estimate the original number of cDNA molecules.
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Figure 1: Schematic of DR-Seq for sequencing gDNA and mRNA from the same cell. (a) gDNA and mRNA/cDNA are shown in red and green, respectively. Following single-cell lysis and RT using adapter Ad-1x (purple), gDNA and single stranded cDNA are amplified by Ad-2 (blue) using a quasilinear amplification strategy. The majority of the short amplicons contain Ad-2 at both ends and cDNA-derived amplicons contain Ad-2 at one end and Ad-1x at the other end. The sample is then split into two halves and processed separately to amplify and sequence gDNA or cDNA. (b) Distribution of reads within 100 nucleotides of the gene Dppa5a for two single cells (red and black) as a function of the random priming location by adapter Ad-2. The unique length-based identifiers found in the two cells can be used to count the original number of cDNA molecules within each cell and minimize amplification biases. The figure shows that distinct positions are randomly primed within each cell with high affinity binding sites being preferentially primed. The size of the dots indicate the binding propensity of each location. For most genes such as Dppa5a, the number of theoretical binding sites far exceed the number of length-based identifiers detected, thereby enabling length-based identifiers to accurately estimate the original number of cDNA molecules.

Mentions: To successfully amplify small quantities of genomic DNA (gDNA) and messenger RNA (mRNA) from single cells in a way that reduces handling, transfer and separation steps, we devised a method (gDNA-mRNA Sequencing, or DR-Seq) that does not involve physical separation of the nucleic acids prior to amplification, thereby minimizing losses and chances of contamination. First, hand-picked single cells are lysed and reverse transcribed using a poly-A primer (called Adapter-1x or Ad-1x) including cell-specific barcodes, a 5′ Illumina adapter and a T7 promoter overhang to convert mRNA to single stranded cDNA (ss cDNA)13 (Fig. 1a). The gDNA and single stranded cDNA are then subjected to quasilinear whole genome amplification, as previously described, using an adapter with a defined 27 nucleotide sequence at the 5′ end followed by 8 random nucleotides (Ad-2)7 (Fig. 1a). After 7 rounds of amplification, the gDNA and cDNA are copied to generate a variety of different short amplicon (0.5–2.5 kb) species, with a majority of amplicons containing adapter Ad-2 at both ends and a small fraction of cDNA derived amplicons containing Ad-2 at one end and Ad-1x at the other (Fig. 1a).


Integrated genome and transcriptome sequencing of the same cell.

Dey SS, Kester L, Spanjaard B, Bienko M, van Oudenaarden A - Nat. Biotechnol. (2015)

Schematic of DR-Seq for sequencing gDNA and mRNA from the same cell. (a) gDNA and mRNA/cDNA are shown in red and green, respectively. Following single-cell lysis and RT using adapter Ad-1x (purple), gDNA and single stranded cDNA are amplified by Ad-2 (blue) using a quasilinear amplification strategy. The majority of the short amplicons contain Ad-2 at both ends and cDNA-derived amplicons contain Ad-2 at one end and Ad-1x at the other end. The sample is then split into two halves and processed separately to amplify and sequence gDNA or cDNA. (b) Distribution of reads within 100 nucleotides of the gene Dppa5a for two single cells (red and black) as a function of the random priming location by adapter Ad-2. The unique length-based identifiers found in the two cells can be used to count the original number of cDNA molecules within each cell and minimize amplification biases. The figure shows that distinct positions are randomly primed within each cell with high affinity binding sites being preferentially primed. The size of the dots indicate the binding propensity of each location. For most genes such as Dppa5a, the number of theoretical binding sites far exceed the number of length-based identifiers detected, thereby enabling length-based identifiers to accurately estimate the original number of cDNA molecules.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374170&req=5

Figure 1: Schematic of DR-Seq for sequencing gDNA and mRNA from the same cell. (a) gDNA and mRNA/cDNA are shown in red and green, respectively. Following single-cell lysis and RT using adapter Ad-1x (purple), gDNA and single stranded cDNA are amplified by Ad-2 (blue) using a quasilinear amplification strategy. The majority of the short amplicons contain Ad-2 at both ends and cDNA-derived amplicons contain Ad-2 at one end and Ad-1x at the other end. The sample is then split into two halves and processed separately to amplify and sequence gDNA or cDNA. (b) Distribution of reads within 100 nucleotides of the gene Dppa5a for two single cells (red and black) as a function of the random priming location by adapter Ad-2. The unique length-based identifiers found in the two cells can be used to count the original number of cDNA molecules within each cell and minimize amplification biases. The figure shows that distinct positions are randomly primed within each cell with high affinity binding sites being preferentially primed. The size of the dots indicate the binding propensity of each location. For most genes such as Dppa5a, the number of theoretical binding sites far exceed the number of length-based identifiers detected, thereby enabling length-based identifiers to accurately estimate the original number of cDNA molecules.
Mentions: To successfully amplify small quantities of genomic DNA (gDNA) and messenger RNA (mRNA) from single cells in a way that reduces handling, transfer and separation steps, we devised a method (gDNA-mRNA Sequencing, or DR-Seq) that does not involve physical separation of the nucleic acids prior to amplification, thereby minimizing losses and chances of contamination. First, hand-picked single cells are lysed and reverse transcribed using a poly-A primer (called Adapter-1x or Ad-1x) including cell-specific barcodes, a 5′ Illumina adapter and a T7 promoter overhang to convert mRNA to single stranded cDNA (ss cDNA)13 (Fig. 1a). The gDNA and single stranded cDNA are then subjected to quasilinear whole genome amplification, as previously described, using an adapter with a defined 27 nucleotide sequence at the 5′ end followed by 8 random nucleotides (Ad-2)7 (Fig. 1a). After 7 rounds of amplification, the gDNA and cDNA are copied to generate a variety of different short amplicon (0.5–2.5 kb) species, with a majority of amplicons containing adapter Ad-2 at both ends and a small fraction of cDNA derived amplicons containing Ad-2 at one end and Ad-1x at the other (Fig. 1a).

Bottom Line: We describe a quasilinear amplification strategy to quantify genomic DNA and mRNA from the same cell without physically separating the nucleic acids before amplification.We show that the efficiency of our integrated approach is similar to existing methods for single-cell sequencing of either genomic DNA or mRNA.Further, we find that genes with high cell-to-cell variability in transcript numbers generally have lower genomic copy numbers, and vice versa, suggesting that copy number variations may drive variability in gene expression among individual cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences), Utrecht, the Netherlands. [2] University Medical Center Utrecht, Cancer Genomics Netherlands, Utrecht, the Netherlands.

ABSTRACT
Single-cell genomics and single-cell transcriptomics have emerged as powerful tools to study the biology of single cells at a genome-wide scale. However, a major challenge is to sequence both genomic DNA and mRNA from the same cell, which would allow direct comparison of genomic variation and transcriptome heterogeneity. We describe a quasilinear amplification strategy to quantify genomic DNA and mRNA from the same cell without physically separating the nucleic acids before amplification. We show that the efficiency of our integrated approach is similar to existing methods for single-cell sequencing of either genomic DNA or mRNA. Further, we find that genes with high cell-to-cell variability in transcript numbers generally have lower genomic copy numbers, and vice versa, suggesting that copy number variations may drive variability in gene expression among individual cells. Applications of our integrated sequencing approach could range from gaining insights into cancer evolution and heterogeneity to understanding the transcriptional consequences of copy number variations in healthy and diseased tissues.

Show MeSH
Related in: MedlinePlus