genotyping  (Qiagen)

 
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    Name:
    REPLI g Single Cell Kit
    Description:
    For highly uniform whole genome amplification WGA from single cells or limited sample material Kit contents Qiagen REPLI g Single Cell Kit 24 WGA rxns 2 to 1000 Cells Input Amount 15 min Hands on Time 8 to 16 hr Reaction Time 40g rxns Yields MDA Technology Tube Format For Highly Uniform Whole Genome Amplification WGA from Single Cells or Limited Sample Includes REPLI g sc Polymerase Buffers and Reagents Benefits WGA from single cell material with complete genome coverage Unbiased amplification of genomic loci due to MDA technology Optimized for use with new technologies including NGS Consistent yields of up to 40 µg average product length 10 kb Novel tool for cancer research stem cell research or metagenomics
    Catalog Number:
    150343
    Price:
    529
    Category:
    REPLI g Single Cell Kit
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    Structured Review

    Qiagen genotyping
    REPLI g Single Cell Kit
    For highly uniform whole genome amplification WGA from single cells or limited sample material Kit contents Qiagen REPLI g Single Cell Kit 24 WGA rxns 2 to 1000 Cells Input Amount 15 min Hands on Time 8 to 16 hr Reaction Time 40g rxns Yields MDA Technology Tube Format For Highly Uniform Whole Genome Amplification WGA from Single Cells or Limited Sample Includes REPLI g sc Polymerase Buffers and Reagents Benefits WGA from single cell material with complete genome coverage Unbiased amplification of genomic loci due to MDA technology Optimized for use with new technologies including NGS Consistent yields of up to 40 µg average product length 10 kb Novel tool for cancer research stem cell research or metagenomics
    https://www.bioz.com/result/genotyping/product/Qiagen
    Average 93 stars, based on 43163 article reviews
    Price from $9.99 to $1999.99
    genotyping - by Bioz Stars, 2020-07
    93/100 stars

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    Images

    1) Product Images from "Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases"

    Article Title: Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-018-0494-2

    Genotyping and immunofluorescence of embryos generated by TALEN-mediated genome engineering. a Schematic overview of the strategy to generate an OCT4-EmGFP knock-in allele. The homologous arms of the donor vector are indicated as LA (839 bp) and RA (1001 bp). PCR primers used for PCR genotyping are shown as arrows in different colors. b PCR products obtained using primers L1-F and L1-R were sequenced. Sequence across the targeted region confirmed the correct fusion of EmGFP to the first exon of OCT4. c EmGFP : PCR genotyping using primers G-F and G-R produced bands of 421-bp fragment in the samples from nine embryos, suggesting the possible insertion or precise knock-in of the EmGFP gene. L1: PCR genotyping using primers L1-F and L1-R produced large products (3259 bp) in seven embryo samples, indicating the EmGFP sequence was integrated. The samples 0806.16C1, 0308 M4 and 0308.B1 only contain the larger product, suggesting either both alleles were targeted, or one allele failed to amplify. S1 and S2: PCR genotyping using primers S1-F and S1-R, S2-F and S2-R produced bands with correct size (1795 bp and 2109 bp) in the samples from the targeted embryos. d Immunostaining of targeted blastocysts using anti-GFP antibody showed a signal in the ICM. Scale bar, 50 μM
    Figure Legend Snippet: Genotyping and immunofluorescence of embryos generated by TALEN-mediated genome engineering. a Schematic overview of the strategy to generate an OCT4-EmGFP knock-in allele. The homologous arms of the donor vector are indicated as LA (839 bp) and RA (1001 bp). PCR primers used for PCR genotyping are shown as arrows in different colors. b PCR products obtained using primers L1-F and L1-R were sequenced. Sequence across the targeted region confirmed the correct fusion of EmGFP to the first exon of OCT4. c EmGFP : PCR genotyping using primers G-F and G-R produced bands of 421-bp fragment in the samples from nine embryos, suggesting the possible insertion or precise knock-in of the EmGFP gene. L1: PCR genotyping using primers L1-F and L1-R produced large products (3259 bp) in seven embryo samples, indicating the EmGFP sequence was integrated. The samples 0806.16C1, 0308 M4 and 0308.B1 only contain the larger product, suggesting either both alleles were targeted, or one allele failed to amplify. S1 and S2: PCR genotyping using primers S1-F and S1-R, S2-F and S2-R produced bands with correct size (1795 bp and 2109 bp) in the samples from the targeted embryos. d Immunostaining of targeted blastocysts using anti-GFP antibody showed a signal in the ICM. Scale bar, 50 μM

    Techniques Used: Immunofluorescence, Generated, Knock-In, Plasmid Preparation, Polymerase Chain Reaction, Sequencing, Produced, Immunostaining

    Related Articles

    Amplification:

    Article Title: STR profiling and Copy Number Variation analysis on single, preserved cells using current Whole Genome Amplification methods
    Article Snippet: .. So, after the amplification with REPLI-g single cell kit, Ampli1™ WGA Kit, DOPlify™ WGA and PicoPLEX® WGA, both the suitability of the amplified DNA for STR genotyping and CNV detection was assessed. .. Experimental design In this study, the performance of four WGA methods on a limited number of cells was examined after 24 hour cell-free DNA BCT® preservation (Fig. ).

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing
    Article Snippet: .. The kits tested were: GenomePlex® Single Cell WGA Kit (which we called DOP-1, Sigma-Aldrich, St. Louis, MO, USA); Silicon Biosystem Ampli™ WGA Kit (DOP-2, Silicon Biosystems, Bologna, Italy); NEB Single Cell WGA Kit (DOP-3, New England Biolabs, Ipswich, MA, USA); Qiagen REPLI-g Mini Kit (MDA-1, Qiagen, Düsseldorf, Germany); Qiagen REPLI-g Single Cell Kit (MDA-2, Qiagen, Düsseldorf, Germany); GE Healthcare illustra GenomiPhi V2 DNA Amplification Kit (MDA-3, GE Healthcare, Little Chalfont, Buckinghamshire, England); and Yikon Genomics Single Cell Whole Genome Amplification Kit (MALBAC, Yikon Genomics, China). ..

    Article Title: Performance of four modern whole genome amplification methods for copy number variant detection in single cells
    Article Snippet: .. Ampli-1 WGA, REPLI-g WGA, DOPlify WGA Cell lysis and amplification was performed, using the Ampli-1 WGA kit (Silicon Biosystems, Castel Maggiore, Italy), the REPLI-g single cell kit (Qiagen Hilden, Germany) or the DOPlify WGA kit (Reproductive Health Science, Thebarton, Australia) as described in the respective manufacturer’s instructions. .. All samples were purified following the manufacturer’s protocol of the Genomic DNA Clean & Concentrator kit (version 1.0.0, Zymo Research, Irvine, USA) with 5X binding buffer.

    Whole Genome Amplification:

    Article Title: STR profiling and Copy Number Variation analysis on single, preserved cells using current Whole Genome Amplification methods
    Article Snippet: .. So, after the amplification with REPLI-g single cell kit, Ampli1™ WGA Kit, DOPlify™ WGA and PicoPLEX® WGA, both the suitability of the amplified DNA for STR genotyping and CNV detection was assessed. .. Experimental design In this study, the performance of four WGA methods on a limited number of cells was examined after 24 hour cell-free DNA BCT® preservation (Fig. ).

    Article Title: Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
    Article Snippet: .. Whole genome amplification and quality control PCR WGA was performed according to the manufacturers’ recommendations using 3 different kits: PCR-based Ampli1 (WG-001-050-R02, Silicon Biosystems), combined MDA-PCR PicoPlex (E2620L, New England Biolabs,), and MDA-based REPLI-g (150343, Qiagen) WGA kits. .. The WGA products after Ampli1 and PicoPlex underwent cleanup with NucleoSpin Gel and PCR Clean-up kit (740609, Macherey-Nagel).

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection
    Article Snippet: .. Whole genome amplification WGA was performed on the extracted DNA by two different technologies, GenomePlex® Single Cell Whole Genome Amplification Kit (WGA4) from Sigma and REPLI-g Single Cell Kit from Qiagen. .. For GenomePlex WGA4 kit: 10 μl of DNA was fragmented for 4 min at 99 °C after adding 1 μl of 10× Single Cell Lysis and Fragmentation Buffer.

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection
    Article Snippet: .. For REPLI-g Single Cell Kit: The standard protocol “Whole genome amplification from genomic DNA using the REPLI-g® Single Cell Kit” from Qiagen, as well as the protocol “increased volume” were both used. .. Briefly, 15 µl template DNA was loaded into a 0.2 ml PCR tube.

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing
    Article Snippet: .. The kits tested were: GenomePlex® Single Cell WGA Kit (which we called DOP-1, Sigma-Aldrich, St. Louis, MO, USA); Silicon Biosystem Ampli™ WGA Kit (DOP-2, Silicon Biosystems, Bologna, Italy); NEB Single Cell WGA Kit (DOP-3, New England Biolabs, Ipswich, MA, USA); Qiagen REPLI-g Mini Kit (MDA-1, Qiagen, Düsseldorf, Germany); Qiagen REPLI-g Single Cell Kit (MDA-2, Qiagen, Düsseldorf, Germany); GE Healthcare illustra GenomiPhi V2 DNA Amplification Kit (MDA-3, GE Healthcare, Little Chalfont, Buckinghamshire, England); and Yikon Genomics Single Cell Whole Genome Amplification Kit (MALBAC, Yikon Genomics, China). ..

    Article Title: Performance of four modern whole genome amplification methods for copy number variant detection in single cells
    Article Snippet: .. Ampli-1 WGA, REPLI-g WGA, DOPlify WGA Cell lysis and amplification was performed, using the Ampli-1 WGA kit (Silicon Biosystems, Castel Maggiore, Italy), the REPLI-g single cell kit (Qiagen Hilden, Germany) or the DOPlify WGA kit (Reproductive Health Science, Thebarton, Australia) as described in the respective manufacturer’s instructions. .. All samples were purified following the manufacturer’s protocol of the Genomic DNA Clean & Concentrator kit (version 1.0.0, Zymo Research, Irvine, USA) with 5X binding buffer.

    Article Title: Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
    Article Snippet: .. WGA was performed using PCR-based Ampli1 (WG-001-050-R02, SiliconBiosystems), combined MDA-PCR PicoPlex (E2620L, NewEngland Biolabs,), and MDA-based REPLI-g (150343, Qiagen) WGA kits according to the manufacturers’ recommendations. .. After DNA yield and quality per WGA kit were estimated, DNA of single cells from each WGA group was used for whole exome NGS on 2 platforms.

    Multiple Displacement Amplification:

    Article Title: Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
    Article Snippet: .. Whole genome amplification and quality control PCR WGA was performed according to the manufacturers’ recommendations using 3 different kits: PCR-based Ampli1 (WG-001-050-R02, Silicon Biosystems), combined MDA-PCR PicoPlex (E2620L, New England Biolabs,), and MDA-based REPLI-g (150343, Qiagen) WGA kits. .. The WGA products after Ampli1 and PicoPlex underwent cleanup with NucleoSpin Gel and PCR Clean-up kit (740609, Macherey-Nagel).

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing
    Article Snippet: .. The kits tested were: GenomePlex® Single Cell WGA Kit (which we called DOP-1, Sigma-Aldrich, St. Louis, MO, USA); Silicon Biosystem Ampli™ WGA Kit (DOP-2, Silicon Biosystems, Bologna, Italy); NEB Single Cell WGA Kit (DOP-3, New England Biolabs, Ipswich, MA, USA); Qiagen REPLI-g Mini Kit (MDA-1, Qiagen, Düsseldorf, Germany); Qiagen REPLI-g Single Cell Kit (MDA-2, Qiagen, Düsseldorf, Germany); GE Healthcare illustra GenomiPhi V2 DNA Amplification Kit (MDA-3, GE Healthcare, Little Chalfont, Buckinghamshire, England); and Yikon Genomics Single Cell Whole Genome Amplification Kit (MALBAC, Yikon Genomics, China). ..

    Article Title: Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
    Article Snippet: .. WGA was performed using PCR-based Ampli1 (WG-001-050-R02, SiliconBiosystems), combined MDA-PCR PicoPlex (E2620L, NewEngland Biolabs,), and MDA-based REPLI-g (150343, Qiagen) WGA kits according to the manufacturers’ recommendations. .. After DNA yield and quality per WGA kit were estimated, DNA of single cells from each WGA group was used for whole exome NGS on 2 platforms.

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing
    Article Snippet: .. The results indicated that SCRS data generated by MDA-2 (MDA using the Qiagen REPLI-g Single Cell Kit) presented higher genome recovery sensitivity than those generated by MALBAC and DOP-PCR with the same sequencing depth. .. SCRS data from DOP-PCR had the lowest amplification bias along the entire genome, as well as high reproducibility and the highest single-cell CNVs detection accuracy ( > 90 %).

    Multiple Annealing and Looping–Based Amplification Cycles:

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing
    Article Snippet: .. The kits tested were: GenomePlex® Single Cell WGA Kit (which we called DOP-1, Sigma-Aldrich, St. Louis, MO, USA); Silicon Biosystem Ampli™ WGA Kit (DOP-2, Silicon Biosystems, Bologna, Italy); NEB Single Cell WGA Kit (DOP-3, New England Biolabs, Ipswich, MA, USA); Qiagen REPLI-g Mini Kit (MDA-1, Qiagen, Düsseldorf, Germany); Qiagen REPLI-g Single Cell Kit (MDA-2, Qiagen, Düsseldorf, Germany); GE Healthcare illustra GenomiPhi V2 DNA Amplification Kit (MDA-3, GE Healthcare, Little Chalfont, Buckinghamshire, England); and Yikon Genomics Single Cell Whole Genome Amplification Kit (MALBAC, Yikon Genomics, China). ..

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing
    Article Snippet: .. The results indicated that SCRS data generated by MDA-2 (MDA using the Qiagen REPLI-g Single Cell Kit) presented higher genome recovery sensitivity than those generated by MALBAC and DOP-PCR with the same sequencing depth. .. SCRS data from DOP-PCR had the lowest amplification bias along the entire genome, as well as high reproducibility and the highest single-cell CNVs detection accuracy ( > 90 %).

    Polymerase Chain Reaction:

    Article Title: Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
    Article Snippet: .. Whole genome amplification and quality control PCR WGA was performed according to the manufacturers’ recommendations using 3 different kits: PCR-based Ampli1 (WG-001-050-R02, Silicon Biosystems), combined MDA-PCR PicoPlex (E2620L, New England Biolabs,), and MDA-based REPLI-g (150343, Qiagen) WGA kits. .. The WGA products after Ampli1 and PicoPlex underwent cleanup with NucleoSpin Gel and PCR Clean-up kit (740609, Macherey-Nagel).

    Article Title: Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
    Article Snippet: .. WGA was performed using PCR-based Ampli1 (WG-001-050-R02, SiliconBiosystems), combined MDA-PCR PicoPlex (E2620L, NewEngland Biolabs,), and MDA-based REPLI-g (150343, Qiagen) WGA kits according to the manufacturers’ recommendations. .. After DNA yield and quality per WGA kit were estimated, DNA of single cells from each WGA group was used for whole exome NGS on 2 platforms.

    Generated:

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing
    Article Snippet: .. The results indicated that SCRS data generated by MDA-2 (MDA using the Qiagen REPLI-g Single Cell Kit) presented higher genome recovery sensitivity than those generated by MALBAC and DOP-PCR with the same sequencing depth. .. SCRS data from DOP-PCR had the lowest amplification bias along the entire genome, as well as high reproducibility and the highest single-cell CNVs detection accuracy ( > 90 %).

    Lysis:

    Article Title: Performance of four modern whole genome amplification methods for copy number variant detection in single cells
    Article Snippet: .. Ampli-1 WGA, REPLI-g WGA, DOPlify WGA Cell lysis and amplification was performed, using the Ampli-1 WGA kit (Silicon Biosystems, Castel Maggiore, Italy), the REPLI-g single cell kit (Qiagen Hilden, Germany) or the DOPlify WGA kit (Reproductive Health Science, Thebarton, Australia) as described in the respective manufacturer’s instructions. .. All samples were purified following the manufacturer’s protocol of the Genomic DNA Clean & Concentrator kit (version 1.0.0, Zymo Research, Irvine, USA) with 5X binding buffer.

    Sequencing:

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing
    Article Snippet: .. The results indicated that SCRS data generated by MDA-2 (MDA using the Qiagen REPLI-g Single Cell Kit) presented higher genome recovery sensitivity than those generated by MALBAC and DOP-PCR with the same sequencing depth. .. SCRS data from DOP-PCR had the lowest amplification bias along the entire genome, as well as high reproducibility and the highest single-cell CNVs detection accuracy ( > 90 %).

    Degenerate Oligonucleotide–primed Polymerase Chain Reaction:

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing
    Article Snippet: .. The results indicated that SCRS data generated by MDA-2 (MDA using the Qiagen REPLI-g Single Cell Kit) presented higher genome recovery sensitivity than those generated by MALBAC and DOP-PCR with the same sequencing depth. .. SCRS data from DOP-PCR had the lowest amplification bias along the entire genome, as well as high reproducibility and the highest single-cell CNVs detection accuracy ( > 90 %).

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    • repli g single cell kit  (Qiagen)
    99
    Qiagen repli g single cell kit
    Whole genome amplification by using GenomePlex (WGA4) and <t>REPLI-g.</t> a Comparison of two whole genome amplification methods: GenomePlex (WGA4) and REPLI-g. b Workflow of GenomePlex (WGA4) and REPLI-g kits. c Whole Genome Amplified DNA products. GenomePlex (WGA4) or REPLI-g WGA was performed on DNA from both fresh and fixed HCT116 cells following the vendor’s manuals. The amplified DNA was checked using agarose gel electrophoresis. d DNA yield comparison. GenomePlex (WGA4) or REPLI-g WGA was performed on both fresh and fixed HCT116 cells following the vendor’s manuals. The amplified DNA amount was measured using the Qubit. (*: standard protocol. **: increased DNA volume protocol)
    Repli G Single Cell Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/repli g single cell kit/product/Qiagen
    Average 99 stars, based on 99 article reviews
    Price from $9.99 to $1999.99
    repli g single cell kit - by Bioz Stars, 2020-07
    99/100 stars
    		  Buy from Supplier

    Image Search Results


    Whole genome amplification by using GenomePlex (WGA4) and REPLI-g. a Comparison of two whole genome amplification methods: GenomePlex (WGA4) and REPLI-g. b Workflow of GenomePlex (WGA4) and REPLI-g kits. c Whole Genome Amplified DNA products. GenomePlex (WGA4) or REPLI-g WGA was performed on DNA from both fresh and fixed HCT116 cells following the vendor’s manuals. The amplified DNA was checked using agarose gel electrophoresis. d DNA yield comparison. GenomePlex (WGA4) or REPLI-g WGA was performed on both fresh and fixed HCT116 cells following the vendor’s manuals. The amplified DNA amount was measured using the Qubit. (*: standard protocol. **: increased DNA volume protocol)

    Journal: NPJ Genomic Medicine

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    doi: 10.1038/s41525-017-0034-3

    Figure Lengend Snippet: Whole genome amplification by using GenomePlex (WGA4) and REPLI-g. a Comparison of two whole genome amplification methods: GenomePlex (WGA4) and REPLI-g. b Workflow of GenomePlex (WGA4) and REPLI-g kits. c Whole Genome Amplified DNA products. GenomePlex (WGA4) or REPLI-g WGA was performed on DNA from both fresh and fixed HCT116 cells following the vendor’s manuals. The amplified DNA was checked using agarose gel electrophoresis. d DNA yield comparison. GenomePlex (WGA4) or REPLI-g WGA was performed on both fresh and fixed HCT116 cells following the vendor’s manuals. The amplified DNA amount was measured using the Qubit. (*: standard protocol. **: increased DNA volume protocol)

    Article Snippet: For REPLI-g Single Cell Kit: The standard protocol “Whole genome amplification from genomic DNA using the REPLI-g® Single Cell Kit” from Qiagen, as well as the protocol “increased volume” were both used.

    Techniques: Whole Genome Amplification, Amplification, Agarose Gel Electrophoresis

    Sequencing comparison in WGA amplified and non-amplified DNA samples. a Mutation detection comparison for REPLI-g WGA. REPLI-g amplified or non-amplified DNA from both fresh and fixed HCT116 cells were subjected to the CRC targeted NGS. The blue color highlights the true mutations detected, while the number inside each cell represents the MAF of the mutation. The light red color highlights the false mutations called. b Gene coverage comparison for WGA. The coverage was compared among the following samples: ① Fresh cells; ② Fixed cells; ③ Fresh cells + REPLI-g; ④ Fixed cells + REPLI-g. ⑤ Fixed cells+WGA4. The reads were aligned using BWA-MEM and coverage was computed using GATK’s DepthOfCoverage and in-house scripts. The percentage of bases covered by at least 10 reads with minimum base quality score Q30 (%_bases_above_10) was calculated using data obtained from DepthOfCoverage and in-house scripts and summarized in the table. (*) In the case of GenomePlex (WGA4), the plotted data was trimmed of 10 bp at the beginning and 40 bp at the end because of a primer/adapter contamination

    Journal: NPJ Genomic Medicine

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    doi: 10.1038/s41525-017-0034-3

    Figure Lengend Snippet: Sequencing comparison in WGA amplified and non-amplified DNA samples. a Mutation detection comparison for REPLI-g WGA. REPLI-g amplified or non-amplified DNA from both fresh and fixed HCT116 cells were subjected to the CRC targeted NGS. The blue color highlights the true mutations detected, while the number inside each cell represents the MAF of the mutation. The light red color highlights the false mutations called. b Gene coverage comparison for WGA. The coverage was compared among the following samples: ① Fresh cells; ② Fixed cells; ③ Fresh cells + REPLI-g; ④ Fixed cells + REPLI-g. ⑤ Fixed cells+WGA4. The reads were aligned using BWA-MEM and coverage was computed using GATK’s DepthOfCoverage and in-house scripts. The percentage of bases covered by at least 10 reads with minimum base quality score Q30 (%_bases_above_10) was calculated using data obtained from DepthOfCoverage and in-house scripts and summarized in the table. (*) In the case of GenomePlex (WGA4), the plotted data was trimmed of 10 bp at the beginning and 40 bp at the end because of a primer/adapter contamination

    Article Snippet: For REPLI-g Single Cell Kit: The standard protocol “Whole genome amplification from genomic DNA using the REPLI-g® Single Cell Kit” from Qiagen, as well as the protocol “increased volume” were both used.

    Techniques: Sequencing, Whole Genome Amplification, Amplification, Mutagenesis, Next-Generation Sequencing

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Journal: NPJ Genomic Medicine

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    doi: 10.1038/s41525-017-0034-3

    Figure Lengend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Article Snippet: For REPLI-g Single Cell Kit: The standard protocol “Whole genome amplification from genomic DNA using the REPLI-g® Single Cell Kit” from Qiagen, as well as the protocol “increased volume” were both used.

    Techniques: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    A narrowing-down strategy used to compare WGA methods cost-effectively. We describe the narrowing-down strategy using 3 panels ( a , b , c ). We perform LWGS comparison including genome coverage and uniform using YH single cells which are amplified by seven WGA kits based on DOP, MDA and MALBAC methods in panel A. We additionally compare the CNVs detection using simulated data of YH single cells in panel A. In panel B, we perform the deep WGS comparison of biases and SNVs detection using deep-sequenced YH or SW480 single cells amplified by DOP, MDA or MALBAC respectively. Corresponding bulk data is used as unamplified control. In panel C, we further compare the CNVs detection between MDA-2 and MALBAC amplified data using real data of BGC823 single cells. *Ion Proton sequencing data; #Illumina and Ion Proton sequencing data. LWGS, low-coverage whole-genome sequencing; WGS: whole genome sequencing. DOP-1,GenomePlex® Single Cell WGA Kit; DOP-2, Silicon Biosystem Ampli ™ WGA Kit; DOP-3, NEB Single Cell WGA Kit; MDA-1, Qiagen REPLI-g Mini Kit; MDA-2, Qiagen REPLI-g Single Cell Kit; MDA-3, GE Healthcare illustra GenomiPhi V2 DNA Amplification Kit; MALBAC,Yikon Genomics Single Cell Whole Genome Amplification Kit. Data marked in purple is downloaded

    Journal: GigaScience

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

    doi: 10.1186/s13742-015-0068-3

    Figure Lengend Snippet: A narrowing-down strategy used to compare WGA methods cost-effectively. We describe the narrowing-down strategy using 3 panels ( a , b , c ). We perform LWGS comparison including genome coverage and uniform using YH single cells which are amplified by seven WGA kits based on DOP, MDA and MALBAC methods in panel A. We additionally compare the CNVs detection using simulated data of YH single cells in panel A. In panel B, we perform the deep WGS comparison of biases and SNVs detection using deep-sequenced YH or SW480 single cells amplified by DOP, MDA or MALBAC respectively. Corresponding bulk data is used as unamplified control. In panel C, we further compare the CNVs detection between MDA-2 and MALBAC amplified data using real data of BGC823 single cells. *Ion Proton sequencing data; #Illumina and Ion Proton sequencing data. LWGS, low-coverage whole-genome sequencing; WGS: whole genome sequencing. DOP-1,GenomePlex® Single Cell WGA Kit; DOP-2, Silicon Biosystem Ampli ™ WGA Kit; DOP-3, NEB Single Cell WGA Kit; MDA-1, Qiagen REPLI-g Mini Kit; MDA-2, Qiagen REPLI-g Single Cell Kit; MDA-3, GE Healthcare illustra GenomiPhi V2 DNA Amplification Kit; MALBAC,Yikon Genomics Single Cell Whole Genome Amplification Kit. Data marked in purple is downloaded

    Article Snippet: The results indicated that SCRS data generated by MDA-2 (MDA using the Qiagen REPLI-g Single Cell Kit) presented higher genome recovery sensitivity than those generated by MALBAC and DOP-PCR with the same sequencing depth.

    Techniques: Whole Genome Amplification, Amplification, Multiple Displacement Amplification, Multiple Annealing and Looping–Based Amplification Cycles, Sequencing

    Read-data CNVs detection comparison between MALBAC and MDA-2 amplified data. a Taking the simulated YH-mix data as control, sensitivity and specificity of CNVs (≥1 Mb) in simulated single YH cells amplified by different WGA methods are bar-plotted. b CNVs of BGC823 single cells amplified by MALBAC or MDA-2. BGC823 single cells are sequenced on the Ion Proton sequencer (~0.5X) as control. Bulk BGC823 sequencing data (bottom row) are sequenced by PE-100 on an Illumina Hiseq 2000 (~50X), and ~1X data was extracted randomly to detect CNVs. Green, red, and blue represent normal, amplification, and deletion, respectively

    Journal: GigaScience

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

    doi: 10.1186/s13742-015-0068-3

    Figure Lengend Snippet: Read-data CNVs detection comparison between MALBAC and MDA-2 amplified data. a Taking the simulated YH-mix data as control, sensitivity and specificity of CNVs (≥1 Mb) in simulated single YH cells amplified by different WGA methods are bar-plotted. b CNVs of BGC823 single cells amplified by MALBAC or MDA-2. BGC823 single cells are sequenced on the Ion Proton sequencer (~0.5X) as control. Bulk BGC823 sequencing data (bottom row) are sequenced by PE-100 on an Illumina Hiseq 2000 (~50X), and ~1X data was extracted randomly to detect CNVs. Green, red, and blue represent normal, amplification, and deletion, respectively

    Article Snippet: The results indicated that SCRS data generated by MDA-2 (MDA using the Qiagen REPLI-g Single Cell Kit) presented higher genome recovery sensitivity than those generated by MALBAC and DOP-PCR with the same sequencing depth.

    Techniques: Multiple Annealing and Looping–Based Amplification Cycles, Multiple Displacement Amplification, Amplification, Whole Genome Amplification, Sequencing

    Bias and chimeras comparison using WGS data. a The cumulative distribution of sequencing fold depth of deep WGS data amplified by DOP-1, MDA-2, MDA-3, and MALBAC, respectively. The standard Poisson Cumulative Distribution (λ = 30) is plotted (dashed), and YH-mix and SW480 bulk data are presented as a control. It was related to Additional file 11 : Table S6. b Normalized read depth distribution in repeat regions (Alu and L1 regions) and the entire genome of deep-sequenced data amplified by different WGA kits. The normalized read depth is calculated for each Alu/L1 region and for each window binning 100 kb of the entire genome. c Normalized read depth distribution in regions with different GC content of deep-sequenced data amplified by different WGA kits. The 100 kb windows with GC content > 50 % are defined as ‘HighGC’ windows,

    Journal: GigaScience

    Article Title: Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

    doi: 10.1186/s13742-015-0068-3

    Figure Lengend Snippet: Bias and chimeras comparison using WGS data. a The cumulative distribution of sequencing fold depth of deep WGS data amplified by DOP-1, MDA-2, MDA-3, and MALBAC, respectively. The standard Poisson Cumulative Distribution (λ = 30) is plotted (dashed), and YH-mix and SW480 bulk data are presented as a control. It was related to Additional file 11 : Table S6. b Normalized read depth distribution in repeat regions (Alu and L1 regions) and the entire genome of deep-sequenced data amplified by different WGA kits. The normalized read depth is calculated for each Alu/L1 region and for each window binning 100 kb of the entire genome. c Normalized read depth distribution in regions with different GC content of deep-sequenced data amplified by different WGA kits. The 100 kb windows with GC content > 50 % are defined as ‘HighGC’ windows,

    Article Snippet: The results indicated that SCRS data generated by MDA-2 (MDA using the Qiagen REPLI-g Single Cell Kit) presented higher genome recovery sensitivity than those generated by MALBAC and DOP-PCR with the same sequencing depth.

    Techniques: Sequencing, Amplification, Multiple Displacement Amplification, Multiple Annealing and Looping–Based Amplification Cycles, Whole Genome Amplification

    Plots of CNA profiles along the whole genome (x axis) ( A ) CNA profile of unamplified DNA from unfixed cells. ( B – G ) plots of CNAs in single SK-BR-3 cells, obtained from EDTA-preserved blood. ( H , I ) CNA profiles of individual CTCs, obtained from EDTA-preserved blood of the same breast cancer patient. WGA kits: (B, E) Ampli1; (C, F, H, I) PicoPlex; (D, G) REPLI-g.

    Journal: Oncotarget

    Article Title: Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells

    doi: 10.18632/oncotarget.10701

    Figure Lengend Snippet: Plots of CNA profiles along the whole genome (x axis) ( A ) CNA profile of unamplified DNA from unfixed cells. ( B – G ) plots of CNAs in single SK-BR-3 cells, obtained from EDTA-preserved blood. ( H , I ) CNA profiles of individual CTCs, obtained from EDTA-preserved blood of the same breast cancer patient. WGA kits: (B, E) Ampli1; (C, F, H, I) PicoPlex; (D, G) REPLI-g.

    Article Snippet: WGA was performed using PCR-based Ampli1 (WG-001-050-R02, SiliconBiosystems), combined MDA-PCR PicoPlex (E2620L, NewEngland Biolabs,), and MDA-based REPLI-g (150343, Qiagen) WGA kits according to the manufacturers’ recommendations.

    Techniques: Whole Genome Amplification

    Distribution of identified known SNPs between datasets ( A ) Known SNPs identified in single cells, amplified with Ampli1, PicoPlex, and REPLI-g WGA kits and obtained from EDTA-preserved blood in comparison to unamplified DNA. ( B ) Known SNPs identified in single cells, amplified with Ampli1 or PicoPlex and obtained from EDTA- and CellSave-preserved blood in comparison to unamplified DNA from unfixed cells. ( C ) Known SNPs identified in single CTCs, amplified with PicoPlex in comparison to each other.

    Journal: Oncotarget

    Article Title: Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells

    doi: 10.18632/oncotarget.10701

    Figure Lengend Snippet: Distribution of identified known SNPs between datasets ( A ) Known SNPs identified in single cells, amplified with Ampli1, PicoPlex, and REPLI-g WGA kits and obtained from EDTA-preserved blood in comparison to unamplified DNA. ( B ) Known SNPs identified in single cells, amplified with Ampli1 or PicoPlex and obtained from EDTA- and CellSave-preserved blood in comparison to unamplified DNA from unfixed cells. ( C ) Known SNPs identified in single CTCs, amplified with PicoPlex in comparison to each other.

    Article Snippet: WGA was performed using PCR-based Ampli1 (WG-001-050-R02, SiliconBiosystems), combined MDA-PCR PicoPlex (E2620L, NewEngland Biolabs,), and MDA-based REPLI-g (150343, Qiagen) WGA kits according to the manufacturers’ recommendations.

    Techniques: Amplification, Whole Genome Amplification