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. 2015 Feb:772:55-62.
doi: 10.1016/j.mrfmmm.2015.01.002.

Single-cell transcriptogenomics reveals transcriptional exclusion of ENU-mutated alleles

Single-cell transcriptogenomics reveals transcriptional exclusion of ENU-mutated alleles

Wenge Li et al. Mutat Res. 2015 Feb.

Abstract

Recently, great progress has been made in single cell genomics and transcriptomics. Here, we present an integrative method, termed single-cell transcriptogenomics (SCTG), in which whole exome sequencing and RNA-seq is performed concurrently on single cells. This methodology enables one to track germline and somatic variants directly from the genome to the transcriptome in individual cells. Mouse embryonic fibroblasts were treated with the powerful mutagen ethylnitrosourea (ENU) and subjected to SCTG. Interestingly, while germline variants were found to be transcribed in an allelically balanced fashion, a significantly different pattern of allelic exclusion was observed for ENU-mutant variants. These results suggest that the adverse effects of induced mutations, in contrast to germline variants, may be mitigated by allelically biased transcription. They also illustrate how SCTG can be instrumental in the direct assessment of phenotypic consequences of genomic variants. VSports手机版.

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Conflict of interest statement

Conflict of Interest statement

The authors declare that there is no conflict of interest.

Figures

Figure 1
Figure 1. Schematic illustration of single-cell transcriptogenomics (SCTG)
After cell lysis, the polyA tailed (AAA) mRNAs (black lines) are selectively pulled down by oligo-dT peptidyl nucleic acid (PNA) beads. After reverse transcription (RT) and terminal deoxynucleotide transferation (TdT), global mRNA amplification is performed by PCR using poly C primers. The resultant cDNA products are subjected to RNA-seq. After alignment to the RefSeq, the cDNAs exhibit discontinued alignment patterns, with discrete exons (solid lines) linked by introns (dashed lines) that have been spliced out. The PNA wash-off fractions containing genomic DNAs (gDNAs, blue lines) are collected, and gDNAs are precipitated, pre-treated with RNaseA and subjected to whole genome amplification (WGA). The WGA products are subjected to whole exome sequencing (WES). The WES and RNA-seq data are subjected to mutation detection analysis. Direct comparison of the mutation profiles from gDNAs and cDNAs reveals the expression of a genomic mutation, such as a heterozygous point mutation (red circles).
Figure 2
Figure 2. Validation of successful WGA and WTA from the same single cell
(A) Agarose electrophoresis indicated the high-molecular weight amplification product expected after successful whole genome amplification (WGA). (B) An example of a locus dropout test (LDO) covering 8 loci on 4 chromosomes indicating successful, relatively unbiased WGA. Numbers 1 to 8 indicate the LDO primer pairs. (C) BioAnalyzer (BA) image indicated successful whole transcriptome amplification (WTA). Samples N1 to M8 were run on one BA High Sensitivity DNA chip, with samples M9 to N2 on another. The two BA runs underwent identical image processing and were combined into one image. (D) Multi-dimensional scaling (MDS) plot confirmed MEF-specificity of single cell WTA amplicons. The MDS plot illustrates that when clustering the top 500 most variable genes between the MEF bulk and a number of publicly available RNA-seq data sets from MEFs and other cells and tissues, single amplified MEFs tended to cluster together with MEF cell populations but not with other types of cells/tissues. Abbreviation: Adip: adipose tissue; CD4+T: CD4+ T cell; CD8+T: CD8+ T cell; E. Heart: embryonic heart; L. Ventricle: left ventricle; M1 to M10: MEF1 to MEF10; MEF: single amplified MEF of the current study; MEF pool: unamplified MEF bulk of the current study; MEF (sra): RNA-seq data of MEF bulk downloaded from sequence read archive; N1: negative control 1 (no reverse transcriptase); N2: negative control 2 (no cell); U: Unamplified control.
Figure 3
Figure 3. Allelic distribution of detected single nucleotide variants (SNVs)
(A) The allelic distribution of the germline SNVs of 7 single MEFs combined. (B) The allelic distribution of germline SNVs in the unamplified, bulk control (C) The allelic distribution of somatic SNVs from 7 single MEFs combined.
Figure 4
Figure 4. Integrative genomics viewer (IGV) images comparing specific mutations present in both exome and transcriptome from the same single cell
The genetic background is defined by the unamplified control (Unamp). (A) The presence of a germline SNV in mRNAs. (B) The presence of a heterozygous somatic A:T transversion, a signature ENU-induced mutation, in mRNAs. (C) A heterozygous somatic SNV absent from mRNAs. (D) A somatic C to A transversion generating a nonsense mutation; no mutant transcript detected. (E) A heterogyzous bi-nucleotidyl CG-deletion absent from mRNAs. (F) The binucleotidyl deletion induces a premature TAG (CTA in the illustration; Sfi1 gene is transcribed in reversed order) stop codon (red arrows) in a downstream exon.

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