Sequencing Single Human and Bacterial Cells at Low Coverage for Aneuploidy, CNV, and Genotyping Applications

Single-cell analysis using PCR and arrays is well established for determining aneuploidy, CNV, and genotyping single cells. NGS of single cells presents many barriers to complete and reproducible analysis, including incomplete genome coverage and irreproducible results. However a surprising number of applications can be successfully executed using partial coverage of the genome as long as the coverage is reproducible. These technical applications include a) aneuploidy and copy number variation determination, b) SNP or other single nucleotide variations in a fraction of the genome, and c) identification of complex populations of cells. These technical applications are important for commercial applications such as pre-implantation genetic screening and diagnosis (PGS and PGD), prenatal diagnostics from single or small numbers of fetal cells in maternal circulation, cancer diagnostics from circulating tumor cells, and identification of infectious disease. In these applications coverage can be compromised as long as the partial coverage is reproducible. We have sequenced human and bacterial cells using a version of the Rubicon PicoPLEX technology that is being developed as a single-cell NGS library kit. This PicoPLEX-scD single-cell NGS prep is as simple as the PicoPLEX WGA kit, which is currently used for microarray and PCR studies and diagnostics from single cells. The PicoPLEX-scD prototype kits were used for the above technical applications. Sequencing quality, genome coverage, and reproducibility were measured in flow sorted and microdissected human cells. To verify that the MiSeq NGS could be used for PGS/PGD applications, as many as 24 single human cells were multiplexed on a single lane. Megabase losses or gains of copy number were reproducibly measured with as few as 200,000 clusters per sample. Partial genotyping and variant identification using single cells were also measured. Finally, single cells were studied in mixtures of other genomes.