Performance of four modern whole genome amplification methods for copy number variant detection in single cells

Deleye, Lieselot; Tilleman, Laurentijn; Plaetsen, Ann-Sophie Vander; Cornelis, Senne; Deforce, Dieter; Nieuwerburgh, Filip Van

doi:10.1038/s41598-017-03711-y

Cited by 58 publications

(45 citation statements)

References 27 publications

(34 reference statements)

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“…demonstrates that multiple displacement amplification (MDA) WGA might be applicable for STR analysis 10 . In contrast, MDA is not considered the most preferable WGA method for CNV analysis 6 . The aim of this study was to assess the performance of four commercially available WGA kits for downstream human identification STR on a B-lymphoblastoid cell line.…”

Section: Introductionmentioning

confidence: 99%

“…REPLI-g Single Cell Kit, PicoPLEX WGA Kit, Ampli1 WGA Kit and DOPlify WGA were compared in parallel for their ability to produce WGA product, adequate for human identification STR analysis. The same WGA methods have been tested before in a similar experimental setup to assess their performance to produce a WGA product, adequate for CNV analysis 6 . REPLI-g single cell kit (Qiagen, Hilden, Germany) is based on this MDA mechanism and has already been shown useful for STR analysis 10 .…”

Section: Introductionmentioning

confidence: 99%

“…In the past, the classical DOP-PCR did not result in reliable CNV or STR analysis 13 . Nevertheless, DOPlify Reaction Kit is an advanced version of this classical DOP-PCR and has recently shown promising results for CNV analysis, with minimal bias introduction 6 . This kit has not yet been tested in the context of STR profiling.…”

Section: Introductionmentioning

confidence: 99%

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Short Tandem Repeat analysis after Whole Genome Amplification of single B-lymphoblastoid cells

Deleye

Plaetsen

Weymaere

et al. 2018

Sci Rep

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To allow multiple genetic analyses on a single cell, whole genome amplification (WGA) is required. Unfortunately, studies comparing different WGA methods for downstream human identification Short Tandem Repeat (STR) analysis remain absent. Therefore, the aim of this work was to assess the performance of four commercially available WGA kits for downstream human identification STR profiling on a B-lymphoblastoid cell line. The performance was assessed using an input of one or three micromanipulated cells. REPLI-g showed a very low dropout rate, as it was the only WGA method in this study that could provide a complete STR profile in some of its samples. Although Ampli1, DOPlify and PicoPLEX did not detect all selected STR markers, they seem suitable for genetic identification in single-cell applications.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Short Tandem Repeat analysis after Whole Genome Amplification of single B-lymphoblastoid cells

Deleye

Plaetsen

Weymaere

et al. 2018

Sci Rep

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“…Therefore, whole genome amplification (WGA) is 71 required to generate sufficient DNA quantities. Several WGA approaches have been reported 72 and each has advantages and disadvantages for different applications [35][36][37][38] but most were 73 optimized for mammalian cell analysis [28,36,[38][39][40][41][42][43][44][45][46][47][48][49]. 74 75 To date, reliable detection of CNV in single P. falciparum parasites using whole genome 76 sequencing has not been achieved.…”

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confidence: 99%

Single cell sequencing of the small and AT-skewed genome of malaria parasites

Liu

Huckaby

Brown

et al. 2020

Preprint

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24Single cell genomics is a rapidly advancing field; however, most techniques are designed for 25 mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, 26 Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed 27 base content. Through optimization of a quasi-linear genome amplification method, we achieve 28 more even genome coverage and better targeting of the parasite genome over contaminants. 29 These improvements are particularly important for expanding the accessibility of single cell 30 approaches to new organisms and cell types and for improving the study of genetic mechanisms 31 of adaptation. 32 33 Keywords: whole-genome amplification, AT-skewed genome, malaria, single cell 34 sequencing, MALBAC 35 36 Background 37 Malaria is a life-threatening disease caused by protozoan Plasmodium parasites. P. falciparum 38 causes the greatest number of human malaria deaths [1]. The clinical symptoms of malaria occur 39 when parasites invade human erythrocytes and undergo rounds of asexual reproduction by 40 maturing from early forms into late stage parasites and bursting from erythrocytes to begin the 41 cycle again [2]. In this asexual cycle, parasites possess a single haploid genome during the early 42 stages; rapid genome replication in the later stages leads to an average of 16 genome copies [2].43 44Due to a lack of an effective vaccine, antimalarial drugs are required to treat malaria. However, 45 drug efficacy is threatened by the frequent emergence of resistant populations [3]. Copy number variations (CNVs), or the amplification and deletion of a genomic element, is one of the major 47 sources of genomic variation in P. falciparum that contribute to antimalarial resistance [4][5][6][7][8][9][10][11][12][13][14][15]. 48Similar to bacteria and viruses [16][17][18], a high rate of CNVs may initiate genomic changes that 49 contribute to the rapid adaptation of this organism [7,19]. Despite the importance of CNVs, their 50 dynamics in evolving populations are not well understood. 52The majority of CNVs in P. falciparum have been identified by analyzing bulk DNA in which 53 the CNVs are present in a substantial fraction of individual parasites in the population due to 54 positive selection [8,10,11,20,21]. However, many CNVs likely remain undetected because 55 they are presumably either deleterious or offer no advantages for parasite growth or transmission 56 [22] and are therefore present in low frequency [20,22]. Currently, most CNVs are identified 57 using read-depth analysis of short read sequencing data, which derives an average signal across 58 the population. For this reason, genetic variants must be present in a high frequency (i.e. ~50%) 59 in the population to be detected [23][24][25]. Sequencing at very high depth improves the detection 60 of low frequency CNVs, but the sensitivity is limited to large-scale CNVs present in > 5% cells 61 [26][27][28]. Analysis methods that rely on the detection of reads that span CNV junctions h...

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“…While several studies comparing the relative performance of different scWGA strategies have already been published, their scope is usually limited in terms of the sequencing target, number of scWGA methods evaluated and/or number and type of amplified cells 16,17,[19][20][21][22][23][24][25][26][27][28][29][30] (Supplementary Table 1). To date, we are not aware of any study comparing a large number of scWGA strategies on whole genomes obtained from a large number of individual cells.…”

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confidence: 99%

Comparison of single-cell whole-genome amplification strategies

Estévez-Gómez

Prieto

Guillaumet-Adkins

et al. 2018

Preprint

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Singlecell genomics is an alluring area that holds the potential to change the way we understand cell populations. Due to the small amount of DNA within a single cell, wholegenome amplification becomes a mandatory step in many singlecell applications. Unfortunately, singlecell wholegenome amplification (scWGA) strategies suffer from several technical biases that complicate the posterior interpretation of the data. Here we compared the performance of six different scWGA methods (GenomiPhi, REPLIg, TruePrime, Ampli1, MALBAC, and PicoPLEX) after amplifying and lowpass sequencing the complete genome of 230 healthy/tumoral human cells. Overall, REPLIg outperformed competing methods regarding DNA yield, amplicon size, amplification breadth, amplification uniformity -being the only method with a random amplification bias-, and false singlenucleotide variant calls. On the other hand, nonMDA methods, and in particular Ampli1, showed less allelic imbalance and ADO, more reliable copynumber profiles and less chimeric amplicons. While no single scWGA method showed optimal performance for every aspect, they clearly have distinct advantages.Our results provide a convenient guide for selecting a scWGA method depending on the question of interest while revealing relevant weaknesses that should be considered during the analysis and interpretation of singlecell sequencing data.

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Performance of four modern whole genome amplification methods for copy number variant detection in single cells

Cited by 58 publications

References 27 publications

Short Tandem Repeat analysis after Whole Genome Amplification of single B-lymphoblastoid cells

Short Tandem Repeat analysis after Whole Genome Amplification of single B-lymphoblastoid cells

Single cell sequencing of the small and AT-skewed genome of malaria parasites

Comparison of single-cell whole-genome amplification strategies

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