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Accurate detection of SNVs and CNVs from five-cell inputs in a single, low-pass sequencing run A single NGS assay for SNVs and CNVs
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Accurate detection of SNVs and CNVs from five-cell inputs in a single, low-pass sequencing run A single NGS assay for SNVs and CNVs

PicoPLEX citations

Our collaborators and customers are constantly making scientific breakthroughs. Here are the latest publications comparing PicoPLEX technology to other single-cell whole-genome amplification technologies, and using PicoPLEX technology to uncover insights from CTCs, single cells from FFPE tumor samples, and samples relevant to preimplantation genetic testing.

Technology comparison studies


Babayan, A. et al. Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells. Oncotarget 8, 56066–56080 (2017).
This paper compared the performance of three commercially available whole genome amplification (WGA) technologies: the PicoPLEX WGA Kit, the REPLI-g Single Cell Kit, and the Ampli1 WGA Kit. Single and pooled tumor cells (SK-BR-3) obtained from EDTA- and CellSave-preserved blood and archival material were whole-genome amplified. The amplified DNA underwent exome capturing, followed by sequencing on both Illumina HiSeq® 2000 and Thermo Fisher Ion Proton platforms. The study concluded that the copy number aberration (CNA) profiles produced with the PicoPLEX WGA Kit were the most accurate and "resembled unamplified DNA the most."

Deleye, L. et al. Performance of four modern whole genome amplification methods for copy number variant detection in single cells. Sci. Rep. 7, 3422 (2017).
This paper compared the performance of the PicoPLEX DNA-seq Kit, the DOPlify WGA Kit, the REPLI-g Single Cell Kit, and the Ampli1 WGA Kit for aneuploidy screening and copy number analysis using shallow whole genome sequencing, starting from one, three, or five cells isolated from the Loucy cell line. Cells were whole-genome amplified, followed by PCR-free library preparation and Illumina sequencing. The PicoPLEX DNA-seq Kit did not require a separate library preparation step as the kit performs whole genome amplification (WGA) and library construction simultaneously, resulting "in a sequencing-ready library, using a single-tube reaction." The study found that PicoPLEX DNA-seq libraries "detected 100% of the CNVs that were also detected in the sequenced bulk sample...leading to the highest number of detected true positives without detection of false positives."

Zhang, X. et al. The comparison of the performance of four whole genome amplification kits on ion proton platform in copy number variation detection. Biosci. Rep. 37, BSR20170252 (2017).
This paper compared the performance of copy number variation (CNV) detection of four commercially available whole genome amplification (WGA) kits: the PicoPLEX WGA Kit, the GenomePlex Single Cell Whole Genome Amplification Kit, the MALBAC Single Cell Whole Genome Amplification Kit, and the REPLI-g Single Cell Kit. The study used six cell lines with different karyotypes and prepared single cells, three to five cells, and 15 pg of isolated gDNA as input for WGA reactions. NGS libraries were constructed for sequencing on the Ion Proton platform. The study concluded that "PicoPLEX showed the best performance in the quality of sequencing data, uniformity of read depth, amplification reproducibility and fidelity," and "recommended [PicoPLEX] for CNV calling on Ion Proton platform."

Konstantinidis, M. et al. Simultaneous assessment of aneuploidy, polymorphisms, and mitochondrial DNA content in human polar bodies and embryos with the use of a novel microarray platform. Fertil. Steril. 102, 1385–92 (2014).
Forty-eight WGA products were produced with the use of MDA, Genomeplex, and PicoPLEX technologies applied to isolated single cells. The PicoPLEX WGA method was determined to be the most successful, and consequently, this was the method used during all subsequent work in this study. Analysis of PicoPLEX WGA products provided a correct diagnosis for 16/16 cells, with detection of 16/16 individual aneuploidies. In contrast, only a minority of aneuploidies was detected in MDA samples and none in the Genomeplex products. In addition, artefactual losses and gains of some chromosomes were observed for MDA and Genomeplex samples.

Genomic profiling of single cells from FFPE tumor tissues


Sho, S. et al. Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens. PLoS One 12, e0170897 (2017).
This paper analyzed alterations in the KRAS gene in pancreas fine needle aspirates (FNAs) using digital PCR (dPCR). Single-cell laser microdissection was utilized to identify the minimal number of tumor cells needed for mutation detection. DNA was first extracted from microdissected FFPE tissues, and then the PicoPLEX WGA Kit was used to perform whole genome amplification. Sanger sequencing was performed on the amplified DNA to confirm the results obtained from dPCR KRAS mutation analysis.

Lu, C. et al. A common founding clone with TP53 and PTEN mutations gives rise to a concurrent germ cell tumor and acute megakaryoblastic leukemia. Cold Spring Harb. Mol. case Stud. 2, a000687 (2016).
This study performed genomic analysis on a patient with a concurrent mediastinal germ cell tumor (GCT) and acute myeloid leukemia (AML) to define the clonal relationship between the two cancers. For the GCT sample, gDNA was extracted from a laser-capture microdissected tumor on the FFPE block of the incisional mediastinal biopsy. For the AML sample, gDNA was isolated from flow-sorted megakaryoblasts from cryopreserved cells banked during the diagnostic bone marrow biopsy. Whole genome amplification was performed on 2 ng of GCT gDNA and 8 ng of AML gDNA, using the PicoPLEX WGA Kit to obtain sufficient DNA for exome-capture hybridization. Whole exome sequencing was performed for variant calling, and Sanger sequencing was used to validate putative calls.

Williamson, S. C. et al. Vasculogenic mimicry in small cell lung cancer. Nat. Commun. 7, 13322 (2016).
This study used single-cell genomic analysis to characterize circulating tumor cells (CTCs) from small cell lung cancer (SCLC) and examine the role of vasculogenic mimicry (VM), i.e., the tendency of tumor cells to form endothelial-like vessels. For copy number aberration (CNA) analysis of CTC patient-derived explants (CDX) of tumor regions with high and low levels of VM, sections were stained for prevalence of VM and cells were obtained via laser capture microdissection (LCM). DNA was extracted and whole-genome amplified using the PicoPLEX WGA Kit, followed by NGS library preparation and sequencing on an Illumina MiSeq® to detect copy number aberrations.

Azad, A. A. et al. Androgen Receptor Gene Aberrations in Circulating Cell-Free DNA: Biomarkers of Therapeutic Resistance in Castration-Resistant Prostate Cancer. Clin. Cancer Res. 21, 2315–24 (2015).
This paper examined whether androgen receptor (AR) gene aberrations detectable in circulating cell-free DNA (cfDNA) are associated with resistance to abiraterone acetate and enzalutamide in metastatic castration-resistant prostate cancer (mCRPC) patients. The cfDNA data was compared to matched metastatic tumor biopsies collected from patients. Approximately 50 cells were isolated from each biopsy using laser capture microdissection and whole-genome amplified using the PicoPLEX WGA Kit. Amplified samples were analyzed using array comparative genomic hybridization to detect copy number aberrations.

Genomic profiling of circulating tumor cells (CTCs)


Morrow, C. J. et al. Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study. Ann. Oncol. 27, 1155–1160 (2016).
This paper generated a patient circulating tumor cell (CTC)-derived explant (CDX) using the CTCs from a non-small-cell lung cancer (NSCLC) patient with advanced metastatic disease. CTCs were enriched and implanted into immunocompromised mice, and the resultant tumors were morphologically, immunohistochemically, and genetically compared with the donor patient's diagnostic specimen. Whole exome sequencing was performed on CDX tumors. For validation of the G340A mutation in the PACRG gene, CTCs were captured from the enrichment filters via laser capture microdissection, extracted for DNA, whole-genome amplified using the PicoPLEX WGA Kit, and finally, Sanger sequenced using locus-specific primers.

Wu, Y. et al. High-Resolution Genomic Profiling of Disseminated Tumor Cells in Prostate Cancer. J. Mol. Diagnostics 18, 131–143 (2016).
This study reported an optimized and robust method to reproducibly detect genomic copy number alterations in samples of two to 40 cells. Disseminated tumor cells (DTCs) were isolated from bone marrow aspirates and whole-genome amplified using the PicoPLEX WGA Kit. The amplified samples were then analyzed using a high-resolution single nucleotide polymorphism (SNP)-array platform and refined computational algorithms. Copy number alterations of DTCs were compared with matched metastatic tumors isolated from the same individual to gain biological insight.

Premasekharan, G. et al. An improved CTC isolation scheme for pairing with downstream genomics: Demonstrating clinical utility in metastatic prostate, lung and pancreatic cancer. Cancer Lett. 380, 144–52 (2016).
This study used fluorescence-activated cell sorting (FACS) in combination with an adhesion matrix (CAM) assay to capture and analyze invasive CTCs from peripheral blood collected from cancer patients. The PicoPLEX WGA Kit was used for whole genome amplification of the captured cells, followed by copy number profiling via array comparative genomic hybridization (aCGH).

Cayrefourcq, L. et al. Establishment and Characterization of a Cell Line from Human Circulating Colon Cancer Cells. Cancer Res. 75, 892–901 (2015).
This paper established cell cultures and permanent cell lines from CTCs from a colon cancer patient and characterized the cells at the genomic, transcriptomic, proteomic, and secretomic levels. For genomic analysis, next-generation sequencing was used to profile copy number variations. Single cells or spheres were whole-genome amplified using the PicoPLEX WGA Kit, followed by library construction and sequencing on a HiSeq™ 2500 instrument.

Whole-genome amplification and aneuploidy screening


Fuchs Weizman, N. et al. Towards improving embryo prioritization: parallel next generation sequencing of DNA and RNA from a single trophectoderm biopsy. Sci. Rep. 9, 2853 (2019).
The authors created a clinically applicable method, called preimplantation genetic testing for aneuploidy and transcriptome (PGT-AT), that prepares a biopsy of 4-6 trophectoderm cells for simultaneous DNA- and RNA-seq to determine the ploidy status and transcriptome profile. In this study, two gold-standard Takara Bio technologies were employed: SMART-Seq chemistry for cDNA library preparation from full-length mRNA and PicoPLEX chemistry for cell lysis, whole genome amplification, and library preparation. In addition to achieving 100% concordance of CNV detection between the standard PGT-A method and the PGT-AT method performed on the same blastocysts, they discovered that pathways regulating gene expression and energy metabolism were downregulated in the euploid samples. The authors expressed confidence that the transcriptome profiling aspect could be safely introduced into the current clinical PGT-A workflow, and doing so could greatly improve our embryo prioritization abilities.

Ho, J. R. et al. Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos. Fertil. Steril. 110, 467–475.e2 (2018).
The authors examined cell-free DNA from spent embryo medium (SEM) to detect ploidy and sex and to determine whether assisted hatching and morphologic grade have an impact on cell-free DNA amount and predictive value. All samples (SEM, trophectoderm biopsies, and whole embryos) were subjected to WGA with PicoPLEX technology followed by NGS-based PGT-A. A cell-free DNA concentration of 63.2 ng/µl was enough to get an accurate ploidy diagnosis, and assisted hatching did not seem to affect cell-free DNA metrics. Based on low concordance rates, the authors conclude that cell-free DNA from spent medium is not ready to replace cellular DNA in PGT-A, but it is a promising tool for noninvasive screening.

Kuznyetsov, V. et al. Evaluation of a novel non-invasive preimplantation genetic screening approach. PLoS One 13, e0197262 (2018).
The authors were the first to assess the suitability of SEM in combination with blastocoel fluid (BF) as a noninvasive PGT tool, and they introduced a new technique for noninvasive collection of BF. PicoPLEX technology was used for WGA of SEM, BF, trophectoderm biopsy, and whole blastocyst samples, followed by NGS-based PGT-A. Relatively high levels of concordance of PGT-A results were obtained for all samples. The authors not only demonstrated high concordance between the noninvasive and invasive samples, but they also remarked that the WGA method on combined SEM + BF samples showed superior amplification rates of 100% over previously reported amplification rates from SEM or BF alone.

Tšuiko, O. et al. Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass. Fertil. Steril. 109, 1127–1134.e1 (2018).
The authors were the first to use high-resolution NGS to compare samples from three compartments of the same blastocyst-blastocoel fluid (BF), trophectoderm (TE), and inner cell mass (ICM)-to determine whether cell-free DNA from BF represents the chromosomal status of the rest of the embryo. Using PicoPLEX technology, DNA from all samples was successfully amplified and then subjected to NGS-based PGT-A. The authors found low concordance (40%) between BF and TE or ICM samples, compared to 86% between TE and ICM. BF contained more mosaic aneuploidies and affected chromosomes than matching TE and ICM samples. They conclude that as a single source of genetic material, BF is not diagnostically acceptable with current protocols, and TE biopsy remains the most effective and safest way of determining karyotype.

Vera-Rodriguez, M. et al. Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Obstet. Gynecol. Surv. 73, 355–356 (2018).
The authors examined the cell-free DNA content of SEM used to culture blastocysts in order to determine cell-free DNA amount and whether a chromosomal diagnosis would be concordant between trophectoderm biopsies and their corresponding culture media samples. NGS-based aneuploidy testing and SNP analysis to detect maternal DNA contamination were performed. PicoPLEX technology was used for double WGA of cell-free DNA in SEM, while FISH was used on the whole blastocysts to detect mosaicism. The cell-free DNA in the media afforded a diagnosis but was discordant with trophectoderm biopsies at a rate of 67%, which was likely due to maternal DNA contamination (92%). Mosaicism was present in most embryos, a possible explanation for the observation of different chromosomal alterations detected between trophectoderm samples and the cell-free DNA from the matching SEM samples.

Vendrell, X. et al. New protocol based on massive parallel sequencing for aneuploidy screening of preimplantation human embryos. Syst. Biol. Reprod. Med. 63, 162–178 (2017).
The authors developed a highly reliable assay for detecting aneuploidy in single blastomeres and samples of 5–10 trophectoderm cells. Their approach-a low-coverage whole genome sequencing method from WGA libraries plus an original algorithm and copy number viewer-depended on PicoPLEX technology to amplify nanogram amounts of gDNA from picogram-level inputs for their NGS protocol. They combined PicoPLEX's limited displacement preamplification with barcodes, followed by PCR amplification, and achieved both a high level of useful reads as well as full correlation of chromosome dose with samples processed using a CGH-BAC microarray approach. They found no differences in sequencing and mapping parameters between unamplified control DNA samples and WGA products from single blastomeres, indicating an unbiased coverage using PicoPLEX technology. Their assay enabled the accurate detection of segmental aneuploidy, with rearrangements of segments down to 1 Mb in length.

Tortoriello, D. V., Dayal, M., Beyhan, Z., Yakut, T. & Keskintepe, L. Reanalysis of human blastocysts with different molecular genetic screening platforms reveals significant discordance in ploidy status. J. Assist. Reprod. Genet. 33, 1467–1471 (2016).
The authors reanalyzed blastocysts that returned abnormal PGT-A results to determine the reliability of different assays run by different labs. They examined the levels of concordance between the first and second rounds of trophectoderm biopsies analyzed by two different PGS labs using aCGH or SNP array (first biopsy/lab) and SNP array or NGS (second biopsy/lab). For NGS, WGA was performed with PicoPLEX technology. Chaotic results were obtained, including normal karyotype or gender-discrepant results upon second biopsy, and it could not be determined whether the lack of concordance was due to mosaicism, differences between platforms, or human error. The authors recommended regular, independent oversight verifying test performance and accuracy.Tšuiko, O. et al. Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass. Fertil. Steril. 109, 1127-1134.e1 (2018).

Vera-Rodríguez, M. et al. Distribution patterns of segmental aneuploidies in human blastocysts identified by next-generation sequencing. Fertil. Steril. 105, 1047–1055.e2 (2016).
The authors evaluated a commonly used NGS-based PGT-A technique for the detection of segmental and whole-chromosome aneuploidies from trophectoderm biopsies and compared these results to the typical aCGH approach. The overall concordance rate between NGS and aCGH was 99.8%, and 92.9% of segments detected by both NGS and aCGH were confirmed by FISH. PicoPLEX technology was used for whole genome amplification, followed by library preparation. The NGS approach detected segmental aneuploidies down to 10 Mb with the same efficiency as aCGH; furthermore, NGS detected several mosaic segmental aneuploidies that were not detected by aCGH. Additionally, the authors determined that most of the segmental aneuploidies resulted from mitotic errors leading to mosaicism, and this mosaic pattern was reliably detected by NGS.

Macaulay, I. C. et al. G&T-seq: Parallel sequencing of single-cell genomes and transcriptomes. Nat. Methods 12, 519–522 (2015).
The authors introduced a new technique, genome and transcriptome sequencing (G&T-seq), which enables a more in-depth characterization of single cells through simultaneous full-length RNA sequencing, whole-genome or targeted sequencing, and genome-wide copy number variation (CNV) detection. Their method involves bead-based isolation of polyA+ mRNA from gDNA, and then separate amplification, library preparation, and sequencing. PicoPLEX technology was chosen to amplify DNA for downstream CNV analysis. The authors produced several interesting and novel findings using their G&T-seq method: 1) chromosomal copy number in a single cell is corroborated by the expected changes in gene expression in that cell; 2) an aneuploidy event that occurs during a single cell division tracks with gene expression dosage; 3) within a single cell, there is high concordance of single nucleotide variation detected in gDNA and mRNA; and 4) a previously unknown fusion and the causative chromosomal rearrangement underlying the fusion were discovered.

Jaroudi, S. & Wells, D. Microarray-CGH for the assessment of aneuploidy in human polar bodies and oocytes. In: Mammalian Oocyte Regulation, 267–283 (Humana Press, Totowa, NJ, 2013).
This paper reviews the use of recent innovations in whole genome amplification and microarray technologies as a means to analyze the copy number of every chromosome in single cells from human oocytes and polar bodies with high accuracy.

Liang, L. et al. Identification of chromosomal errors in human preimplantation embryos with oligonucleotide DNA microarray. PLoS One 8, e61838 (2013).
This study investigated whether the NimbleGen oligonucleotide microarray platform can be used for accurate aneuploidy screening during preimplantation genetic screening of human embryos. Blastocysts from donors of advanced maternal age or with previous miscarriage were analyzed for chromosomal abnormalities using the oligo microarray platform and the PicoPLEX WGA Kit for whole genome amplification, and compared to the traditional FISH (fluorescence in situ hybridization) method. Using the DNA microarray method, a 58.1% rate of chromosomal abnormalities, including copy number variation (CNV) and minor abnormalities, was detected with high sensitivity and reproducibility.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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