Four things to know before you start your single cell DNA analysis

Successful analysis of single cell genome starts with an appropriate Whole Genome Amplification step and ends with an optimized sequencing process which matches the characteristics of the adopted WGA. Going through the following 4 points will help you make an educated choice of the most appropriate workflow for a successful analysis of your single cell: 1. Different WGA methods exist in the market, do you know them all? 2. Are you treating real single-cells or just limited input DNA? 3. What type of genetic analysis are you interested in? 4. Are you planning to use fresh or fixed cells? And once...

Four things to know before you start your single cell DNA analysis Proteinase K digestion of single cell Restriction Enzyme digestion of DNA Fig. 1. Ampli1™ Kit procedure begins with a restriction digestion, which generates exactly 19,046,047 fragments from each of the two haploid genomes, with the same 2-base sticky-ends on both sides. An adaptor, complementary to the sticky ends generated, is ligated to both sides of each fragment and is used, after filling the ends, as a priming site for a single universal PCR primer. This ensures that, regardless of where a mutation is located in the genome,...

Single cell or limited-cell number When working with a limited amount of DNA, which is still the equivalent of tens to hundreds of cells, randomly occurring amplification bias introduced by the WGA process on a single DNA molecule is compensated by a comparable bias, on the opposite direction, on other molecules. The more DNA molecules are present in the sample for each allele, the more likely the average final amplified amount of DNA will accurately represent the original allelic ratio present in the sample, even when a random or semi-random based process is used for the WGA. When working with...

Four things to know before you start your single cell DNA analysis When analyzing haploid cell genomics, the events of interest can be: • Single nucleotide polymorphisms (SNPs); • Copy number variations (CNVs). In this case, the only possible sources of errors induced by the WGA process are polymerase fidelity and the uniformity of representation of the amplified genome. Diploid cells genomic analysis is much more complex, involving: • Homozygous SNPs and insertions/deletions (INDELs); • Heterozygous SNPs and INDELs; • Copy Number Variation (CNVs); • Loss of heterozygosity (LOH). All such determinations...

Fresh vs fixed cell While working with culture cells makes it relatively simple to manage fresh samples, dealing with real patients’ samples is challenging when attempting to collect, transfer and store blood while maintaining cell integrity. Fixation makes the cell stable but induces DNA cross-linking and fragmentation, which heavily affects the efficiency of MDA-based WGA methods, compromising the overall amount of DNA obtained and the uniformity of distribution across the genome. Ampli 1™ WGA kit is the least affected by DNA cross-linking and therefore is the method of choice to obtain large...

Four things to know before you start your single cell DNA analysis Supported protocols Single cell NGS analysis is used to profile copy number aberrations, identify known cancer hotspot mutations and focal CNVs or discover new somatic variation through whole genome/whole exome sequencing. All these protocols require the preparation of an NGS library and the use of appropriate bioinformatics analysis. Ampli1™ WGA Kit workflow includes supported solutions for seamless library generation and data analysis for Illumina and Ion Torrent NGS technologies (Fig. 2). Ampli 1™ QC kit for use with Ampli...

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