Unknown,Transcriptomics,Genomics,Proteomics

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Whole Genome Amplification – surveying yield, reproducibility and heterozygous balance, reported by MIPs targeting STRs

ABSTRACT: Whole Genome Amplification (WGA) is a crucial first step to nearly all single cell genomic analyses with following steps focusing solely on its products. Therefore, bias and variance caused by the WGA process add numerous challenges to the world of single cell genomics. Short Tandem Repeats (STRs) are sensitive genomic markers used widely in population genetics, forensics and retrospective lineage tracing. By extending a previous evaluation of common WGA kits(T, O et al. 2021) targeting ~1000 STR loci across the male X chromosome, to ~12,000 across the entire genome via a DuplexMIPS system, heterogeneous STR loci can be discovered in abundance, and better used to analyze the efficacy and effectiveness of various WGA kits. We show that while best overall yield is obtained using RepliG-SC, maximum uniformity between alleles and reproducibility across cells is maximized by Ampli1, rendering it as the best candidate for comparative heterozygous analysis of SC genomes.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: ehud shapiro

PROVIDER: E-MTAB-11711 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Insufficient quantities of human genomic DNA are a limiting factor for many clinical applications. Whole genome amplification (WGA) is an approach designed to overcome small amount of DNA for genome-wide genetic tests as it allows amplification of the entire genome from picogram or nanogram quantities of DNA. Various strategies of WGA have been developed; however, none of them can guarantee the absence of amplification bias. High-quality genome-representative amplified DNA is crucial for WGA use in basic research and clinical genetics. Thus, systematic evaluation of WGA effect on downstream methods is necessary. Results: In this paper, 4 multiple displacement amplification (MDA) -based and 2 PCR-based WGA kits were compared in their effect on segmental copy-number changes as well as copy-number neutral loss of heterozygosity detection by high-density oligonucleotide DNA arrays. We described outcomes and limits for each individual WGA; however, the main goal of this study was chiefly to show a general compatibility and features specific for particular WGA strategy. The main outcomes are as follows: 1) MDA-based WGAs showed higher tendency to generate false positive imbalances in contrast to PCR-based WGAs with higher risk of false negativity; 2) the specific risk of false positivity and/or negativity increased with decreasing copy-number segments size; 3) single-cell WGAs showed significantly worse effect on results in comparison to WGAs with nanogram level of DNA as input; 4) PCR-based WGAs were not compatible with copy-number neutral loss of heterozygosity analysis based on single nucleotide polymorphisms in restriction digestion sites and also showed higher risk of copy-number neutral loss of heterozygosity false negativity if combined with analysis based on simple hybridization. Conclusions: This study gives a comprehensive insight into the WGA effect on DNA array analysis. The results of this study help to choose WGA according to individual user requirements and options. Moreover, we show a strategy to verify and validate segmental copy-number changes detection by DNA array protocol including any WGA for any purpose to attain the highest efficiency without an unnecessary WGA bias.

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Whole Genome Amplification – surveying yield, reproducibility and heterozygous balance, reported by MIPs targeting STRs

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