Single Cell analysis for DNA-Seq

[Jane M]

Hello everybody,

I will get targeted single cell DNA data in the upcoming days. I have never analysed single cell data, so I searched on this forum and on internet for information, but I haven't been able to find a lot of things for DNA. My goal will be to identify short mutations (already identified by WES and check using PGM).

My question is the following: can we analyse this type of data as it is commonly done for WES, WGS and targeted data (obtained from PGM or MiSeq instruments - without removal of PCR duplicates)?

I would also appreciate other information, like typical alignment rate or average quality,...

Thank you in advance for your help,
Jane

[alec]

If you are looking to test whether mutations identified by WES are present in your single cells, you should be able to use standard SNP calling tools, although you might do better by doing a pileup at those locations and setting a threshold. You didn't specify how you are doing the whole genome amplification, but with any method you should expect a fairly high allele dropout rate--cases in which the cell contains a heterozygous mutation that isn't detected. You can test for this by checking what fraction of known heterozygous SNPs are detected by your algorithm.

If you want to identify new mutations that weren't in your WES data, this becomes much more difficult. The main challenge is that there will be thousands of false positives due to errors by the polymerase during amplification.

For metrics like alignment rate, we recently had a review paper that compares the performance of WGA different kits: http://www.annualreviews.org/doi/abs...-090413-025352

[raimotanzi]

I suggest you use Ampli1 WGA (Menarini Silicon Biosystems) if you are interested in mutation detection on single cells. Several papers report it has an ADO lower than 10%, compared to other WGA >30%.
PLOS ONE | DOI:10.1371/journal.pone.0171566 February 16, 2017