NextSeq 500 paired end sequencing; Comparison of seven single cell Whole Genome Amplification commercial kits using targeted sequencing
Comparison of seven single cell Whole Genome Amplification commercial kits using targeted sequencing
Followed by ~2 weeks of growth, a single clone was selected and detached to enable selection and picking of single cells. For the cell picking procedure cells were treated as described in (Biezuner et al. Genome Research. 2016) with the exception that prior to picking to scWGA reactions, cells were detached and dissociated using 550 U/ml StemPro Accutase Cell Dissociation Reagent (Gibco), incubated for 6 minutes, pulled down, re-suspended in iMEFs Conditioned hESC medium (CM) and were spread over a 6cm non-adherent petri dish.At the day of the experiment, before cell treatment, in order to minimize contamination, a clean working environment was established: bench and pipettes were RNase and DNA decontaminated (RNase AWAY, Molecular BioProducts) and consumables (PCR plates, tubes, tips etc.), non-kit reagents (PCR grade water, buffers etc.) and pipettes were UV irradiated. Individual cells were picked and deposited in separated single wells in dedicated 96-well PCR plates (Axigen, each kit in a separate plate), containing the required reagent. All pickings were performed on the same clone and at the same day (besides GPHI-SC, which was performed a few days later due to technical reasons). 1µl of positive (100pg Hela genomic DNA, NEB) and negative (water) controls were added as templates to non-deposited reaction wells. Samples were directly processed by their appropriate scWGA protocol. All reagents were immediately or sequentially (depending on the protocol) added to the original picking well, without material transfer, to avoid material loss. H1 human ES cells (WA01) were obtained from the WiCell Research Institute (Madison, WI). In order to create single cell (SC) clones, SCs were picked and deposited in separated single 96 wells using an automated cell picking device (CellCelector, ALS). Cells were cultured and treated as described in (Biezuner et al. Genome Research. 2016) The following scWGA kits were used (Kit short in manuscript, Kit name, Cat. Number, WGA technique, Manufacturer): <br> Ampli1, Ampli1 WGA Kit, WG 001 050 R02, Linker adapter PCR (LA-PCR), Silicon Biosystems<br>MALBAC, MALBAC Single Cell WGA Kit, YK001B, Multiple Annealing and Looping Based Amplification Cycles (MALBAC), Yikon Genomics<br>GenomePlex, GenomePlex Single Cell Whole Genome Amplification Kit (WGA4), WGA4, Degenerate oligonucleotide-primed PCR (DOP-PCR), Sigma<br>PicoPlex, PicoPLEX WGA Kit, E2620S, displacement DOP-PCR (D-DOP-PCR), New England Biolabs (manufactured by Rubicon)<br>GPHI-SC, illustra Single Cell GenomiPhi DNA Amplification Kit, 29-1081-07, Multiple Displacement amplification (MDA), GE Healthcare<br>RepliG-SC, REPLI-g Single Cell Kit, 150345, MDA, Qiagen<br>TruePrime, TruePrime Single Cell WGA kit, 350025, MDA, SYGNIS Targeted enrichment using Access Array (Fluidigm) using the protocol as described in (Biezuner et al. Genome Research. 2016.) Sequencing primers are described in manuscript Supplementary Table 1.