Single-cell whole-genome amplification technique impacts the accuracy of SNP microarray-based genotyping and copy number analyses

Treff, Nathan R.; Su, Jing; Tao, Xin; Northrop, Lesley E.; Scott, Richard T.

doi:10.1093/molehr/gaq103

Cited by 92 publications

(69 citation statements)

References 59 publications

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“…The Sureplex WGA procedure consists of three steps: extraction and fragmentation of the genomic DNA of the isolated cells to fragments in the range of a few hundred basepairs, ligation of specific adaptor sequences to both ends of the fragments and a subsequent amplification reaction by PCR using the flanking universal priming sites contained in the adaptors. This system is widely used in the clinical setting for pre-implantation genetic diagnosis starting from single-cell material 34 and, although it only amplifies a representation of the genome, has a relatively low allele-dropout rate and amplification bias 22,23 .…”

Section: Methodsmentioning

confidence: 99%

“…Over the years, many methods for WGA have been developed, each of them with their strengths and weaknesses 22,23 . Mainly, WGA methods differ in their yields and, more importantly, in their amplification bias.…”

Section: Detection Of Genetic Imbalances In Single Cells By Acghmentioning

confidence: 99%

“…This method is known to perform well for the linear amplification of single cells. It generates fragments from 100 to 1,000 base pairs, with an average of 400 basepairs 22 , although it only amplifies a representation of the genome 23 . Since the abnormalities we are calling are in the Mb range, this minimizes the chance for a preferential over-or under-amplified fragment to be misinterpreted as a CNV.…”

Section: Detection Of Genetic Imbalances In Single Cells By Acghmentioning

confidence: 99%

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Low-grade chromosomal mosaicism in human somatic and embryonic stem cell populations

et al. 2014

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Current knowledge on chromosomal mosaicism in human cell cultures is mostly based on cytogenetic banding methods. The recent development of high-resolution full-genome analysis methods applicable to single cells is providing new insights into genetic and cellular diversity. Here we study the genetic content of 92 individual human cells, including fibroblasts, amniocytes and embryonic stem cells (hESCs), using single-cell array-based comparative genomic hybridization (aCGH). We find that human somatic and embryonic stem cell cultures show significant fractions of cells carrying unique megabase-scale chromosomal abnormalities, forming genetic mosaics that could not have been detected by conventional cytogenetic methods. These findings are confirmed by studying seven clonal hESC sub-lines by aCGH. Furthermore, fluorescent in situ hybridisation reveals an increased instability of the subtelomeric regions in hESC as compared to somatic cells. This genetic heterogeneity may have an impact on experimental results and, in the case of hESC, on their potential clinical use.

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Section: Methodsmentioning

confidence: 99%

Section: Detection Of Genetic Imbalances In Single Cells By Acghmentioning

confidence: 99%

Section: Detection Of Genetic Imbalances In Single Cells By Acghmentioning

confidence: 99%

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Low-grade chromosomal mosaicism in human somatic and embryonic stem cell populations

et al. 2014

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“…The fact that any heterozygous calls in the 2nd polar body were observed at all (particularly in those that were euploid) is at least indicative of the former. However, this is not unexpected given the previously identified accuracy limitations of single cell genotyping after whole genome amplification and microarray analysis [7].…”

Section: Discussionmentioning

confidence: 84%

Polar body morphology is not predictive of its cell division origin

Treff

Scott

et al. 2011

J Assist Reprod Genet

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Purpose This study sought to determine whether morphology of simultaneously biopsied polar bodies is predictive of their cell division origin. Methods Levels of heterozygosity were measured using single nucleotide polymorphism (SNP) microarrays in sequentially biopsied polar bodies to establish the predictive value on samples with known cell division origins. The validated method of predicting cell division origin of polar bodies using heterozygosity rates was then applied to simultaneously biopsied polar bodies which had origin predictions made by morphological assessment.Results SNP microarray heterozygosity analysis was proven to be 94% predictive when tested against the known origin of sequentially biopsied polar bodies (n=133). This methodology subsequently demonstrated that morphology was only 63% consistent when tested on simultaneously biopsied polar bodies (n=455; P<0.0001). Predictions of the origins of aneuploidy using morphology assignment were also significantly different than with heterozygosity assignment of polar body cell division origin (P=0.0003). Conclusions Studies of the origin of aneuploidy utilizing morphological assignment of polar body cell division origin without heterozygosity analysis may be inaccurate and should be interpreted with caution.

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“…This is because the underlying copy-number state of the SNP is ignored and because the abundant WGA artifacts in single-cell assays produce false homozygous and heterozygous SNP calls. 13,14 Various methods for DNA copy-number profiling of single cells have been developed and rely on transforming probe intensities of microarrays 3,10,[15][16][17] or next-generation sequence read counts [18][19][20][21] into DNA copy numbers.…”

Section: Introductionmentioning

confidence: 99%