Capítulo 3. El estudio del caso
3.3 Los actos delictivos
3.3.2 Los robos
To demonstrate the system performance and utility for single-cell genomic research, we conducted single-cell mRNA-Seq whole transcriptome analysis for four types of cultured cell lines (K562, BJ Fibroblast, HL-60 and keratinocytes). The results showed >98% mapped reads within 2-fold of the average, >65% mapping rate to RefSeq, <2% mapping rate to rRNA anduniform coverage across transcripts with <4 kb length. The single-cell mRNA-Seq reveals a broad spectrum of transcriptional heterogeneity within nominally homogeneous cell populations of the same cell line in tissue culture, and even greater difference between different cell lines. Further, we observed unique gene expression signatures of the common genes in different types of cells.
$ $%( , ,%( 2 2%( - $ ( ,$ ,( 2$ 2( ) 8 = D (. " ' 8 ;= #8 (% E " FD (. G = 1 #8 - .8FF 567", 567"2 567"- 567"0
No call captured or dead cells
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Figure 6. Sequence read coverage and reproducibility of the single-cell mRNA-Seq workflow. Cultured
K562 cells were applied to 4 C1 IFCs on 4 different days. Twenty four of 96 harvest samples from each IFC were sequenced on MiSeq-V1 with pair end reads (2x30 cycles), resulting in over 4x105 total reads per cell.
Read coverage of 92/93 (>98%) live cells captured is within 2-fold of average. Libraries prepared from “no cell captured “and dead cells as determined by live/dead staining yielded very low read numbers.
Figure 7. Mapping rate of single-cell mRNA-Seq. Cultured K562 cells were applied to 4 C1 IFCs on 4
different days. Twenty-four of 96 harvest samples from each array were sequenced on MiSeq-V1 with pair end reads (2x30 cycles), resulting in over 4x105 total reads per cell. Empty capture sites (“no cell”) and
dead cells are excluded from the plot.
Figure 8. Examples of the transcript length-normalized coverage across all genes with >10 RPKM. A library of 3 individual K562 cells was sequenced on one lane of a HiSeq 2000 Sequencer,
yielding >30 million reads per cell. The position 0 is the 5’-end of the transcripts and 100 is the extreme 3’-end of the transcripts.
Figure 9. Principal component analysis (PCA) of single-cell gene expression.
Data were collected for four types of cultured cells (HL-60, K562, BJ Fibroblast and Keratinocyte; 15 individual cells per cell type). Genes with <10 RPKM are excluded. A total of 12,163 genes were used for the PCA.
prove fruitful, then there is the possibility that thousands of more individuals will require sequencing, which is costly, hence why collaborative initiatives such as UK10K and NHLBI Exome Sequencing Project have been set up. If one wants to move away from rare variant searching involving larger sample sizes, studies involving single cell genomics, with an aim of focusing on gene expression and specific gene interactions within cells and linking them with disease phenotype can provide a closer look at the functional consequences of mutations in certain cell types. This will develop a better understanding of genetic heterogeneity within cells and its relation to disease.
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