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cDNA Synthesis & Library Construction

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Recent Publications using SMARTer PicoPLEX Technology

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

Technology comparison studies

  1. Deleye et al. Performance of four modern whole genome amplification methods for copy number variant detection in single cells. Scientific Reports (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 single, 3, or 5 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."

  2. Zhang et al. The comparison of the performance of four whole genome amplification kits on Ion Proton platform in copy number variation detection. Bioscience Reports (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, 3 to 5 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."

  3. Babyan et al. Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells. Oncotarget (2016).

    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."

Genomic profiling of single cells from FFPE tumor tissues

  1. Sho et al. Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens. PLOS One (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.

  2. Willilamson et al. Vasculogenic mimicry in small cell lung cancer. Nature Communications (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)—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.

  3. Lu et al. A common founding clone with TP53 and PTEN mutations gives rise to a concurrent germ cell tumor and acute megakaryoblastic leukemia. Molecular Case Studies (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.

  4. Azad et al. Androgen Receptor Gene Aberrations in Circulating Cell-Free DNA: Biomarkers of Therapeutic Resistance in Castration-Resistant Prostate Cancer. Clinical Cancer Research (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)

  1. Morrow et al. Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study. Annals of Oncology (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.

  2. Wu et al. High-resolution genomic profiling of disseminated tumor cells in prostate cancer. J. Molecular Diagnostics (2016).

    This study reported an optimized and robust method to reproducibly detect genomic copy number alterations in samples of 2 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.

  3. Premasekharan et al. An improved CTC isolation scheme for pairing with downstream genomics: Demonstrating clinical utility in metastatic prostate, lung and pancreatic cancer. Cancer Letters (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).

  4. Cayrefourcq et al. Establishment and Characterization of a Cell Line from Human Circulating Colon Cancer Cells. Cancer Research (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.

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