Single Cell Genomics: Advances and Future Perspectives (original) (raw)
Figure 1
Detection of various classes of genetic variation using single-cell WGA-NGS approaches.
A) The most prominent methods for (i–ii) isolating individual cells (including (i) creation of single-cell suspensions—usually by enzymatic tissue disaggregation—and subsequent cell isolation through manual micro-pipetting [37], [38], [57], [105], fluorescence-activated cell sorting [106], [107] or microfluidics devices [18], [81], [108], and (ii) laser capture microdissection [109], [110]) as well as (iii) isolating single nuclei [12], [32], [56], [111] are indicated, accompanied with particular advantages and disadvantages. A comprehensive review of single-cell isolation methods is presented by Shapiro et al. [112]. B–D) Subsequently, the cell is lysed and its genome amplified. A standard sequencing library can be prepared from the WGA product for paired-end (or single-end) sequencing. The resulting (short) sequence reads of the cell are mapped against a reference genome for variant discovery (E_i_–E_iii_). In all steps (E_i_–E_iii_ towards F), various confounding factors resulting from the WGA process have to be considered in the analysis (indicated in red boxes). E_i_–F) Structural variants can be detected by analysing read-pairs which map discordantly to the reference genome, or by discovering split reads crossing a rearrangement. However, WGA can create various chimeric DNA molecules that resemble structural variants following paired-end sequence analysis of the WGA-product. E_ii_–F) Copy number variants are called by “binning” reads that map to particular regions of the genome. By comparing the read count per bin to the counts obtained in a reference sample [17], or an average read count per bin [32], a copy number profile can be calculated. However, single-cell copy number profiles can be distorted by ADO, PA, and %GC-bias during the WGA process. E_iii_–F) Single nucleotide variants (SNVs) can be detected in sequenced single-cell WGA products by aligning the reads with a reference genome. However, three cells carrying the same SNV are required to confidently call the variant.