genotyping  (Qiagen)


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    Name:
    REPLI g Screening Kit
    Description:
    For high throughput manual or automated whole genome amplification from small or precious samples Kit contents Qiagen REPLI g Screening Kit 200 x 40L rxns 40L Reaction Volume Multiple Displacement Amplification Technology 8g Yield 10 to 16 hr Reaction Time For High throughput Manual or Automated Whole Genome Amplification from Small or Precious Samples Ideal for Genotyping Sequencing End point PCR Quantitative Real time PCR Microarray Includes DNA Polymerase Buffers and Reagents Benefits Process 96 samples with just 20 minutes of handling time Parallel WGA of multiple samples Highly stable DNA suitable for long term storage Room temperature setup allows easy liquid handl
    Catalog Number:
    150126
    Price:
    1378
    Category:
    REPLI g Screening Kit
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    Structured Review

    Qiagen genotyping
    REPLI g Screening Kit
    For high throughput manual or automated whole genome amplification from small or precious samples Kit contents Qiagen REPLI g Screening Kit 200 x 40L rxns 40L Reaction Volume Multiple Displacement Amplification Technology 8g Yield 10 to 16 hr Reaction Time For High throughput Manual or Automated Whole Genome Amplification from Small or Precious Samples Ideal for Genotyping Sequencing End point PCR Quantitative Real time PCR Microarray Includes DNA Polymerase Buffers and Reagents Benefits Process 96 samples with just 20 minutes of handling time Parallel WGA of multiple samples Highly stable DNA suitable for long term storage Room temperature setup allows easy liquid handl
    https://www.bioz.com/result/genotyping/product/Qiagen
    Average 99 stars, based on 26619 article reviews
    Price from $9.99 to $1999.99
    genotyping - by Bioz Stars, 2020-04
    99/100 stars

    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

    Clone Assay:

    Article Title: Filtering "genic" open reading frames from genomic DNA samples for advanced annotation
    Article Snippet: DNA was amplified by multiple displacement amplification (MDA) with a Repli-g screening kit (Qiagen) according to the manufacturer’s instructions. .. Fragments were twice blunt-ended (Quick blunt kit, NEB) and gel-purified (Gel extraction kit, Qiagen) before cloning into the EcoRV cleaved POS, SOS, and TOS vectors.

    Amplification:

    Article Title: FUS GENE MUTATIONS IN FAMILIAL AND SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
    Article Snippet: .. - The FALS DNA samples were subsequently whole genome amplified in triplicate using the REPLI-g kit (Qiagen, Valencia, CA, USA). .. For all FALS and SALS patients, PCR of FUS exons 5, 6, 14 and 15 was performed with Qiagen products using primers located in adjacent introns.

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: Next, an amplicon covering 8 mouse Htr3a promoter CpGs (located from 96 bp upstream of the transcription start site [TSS] to 151 bp downstream of the TSS) ( ) was generated by the polymerase chain reaction (PCR) using a pair of tagged primers (Forward: aggaagagagGGGGTTGTTAGTATGGAAAGGAATA; Reverse: cagtaatacgactcactatagggagaaggctCCTAACATCTACCAATAAACTCCCC). .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN).

    Article Title: PIK3CA hotspot mutations in circulating tumor cells and paired circulating tumor DNA in breast cancer: a direct comparison study
    Article Snippet: .. Amplification with REPLI‐g kit (Qiagen) was performed according to the manufacturer's recommendations. ..

    Article Title: Filtering "genic" open reading frames from genomic DNA samples for advanced annotation
    Article Snippet: .. DNA was amplified by multiple displacement amplification (MDA) with a Repli-g screening kit (Qiagen) according to the manufacturer’s instructions. .. After 16 h amplification, DNA was fragmented using nitrogen gas based nebulisation for 1 min at 45 psi; average fragment size was around 500 bp (200-800 bp range).

    Article Title: Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements
    Article Snippet: One aliquot of each digested genomic DNA (20ng) was subjected to whole genome amplification respectively using REPLI-G kit (Qiagen) with 8 hours incubation at 30C. .. The amplified DNA sample was then purified by QIAEX II kit (Qiagen) with slightly modified protocol (3 instead of 2 washes with PE buffer and finally eluted in water rather than EB buffer).

    Article Title: Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model
    Article Snippet: .. Whole genome amplified (WGA) genomic DNA, which is completely unmethylated, was created using the Repli-g kit (Qiagen, Valencia, CA) following the manufacturer's protocol and was used as a negative control for MSP assays. .. The resulting DNA was sodium bisulfite modified as previously described [ ].

    Article Title: Opioid Receptor Polymorphism A118G Associated with Clinical Severity in a Drug Overdose Population
    Article Snippet: .. Whole genome amplification was performed using the Qiagen REPLI-g kit from approximately 5 ng of initial genomic DNA per sample, with cycling and hybridization conditions set according to the manufacturer's instructions to produce approximately 1–2 μg amplified DNA per sample. .. The OPRM1 polymorphisms (rs1799971 and rs2075572) were genotyped by the Taqman SNP Genotyping assay (Applied Biosystems, Foster City, CA, USA) by a blinded study investigator.

    Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes
    Article Snippet: Due to limited amounts of starting DNA, we employed “Quantitative Methylation Analysis of Minute DNA Amounts After Whole Bisulfitome Amplification”, known as qMAMBA [ , ]. .. DNA Whole Genome Amplification (WGA) was carried out on bisulfite-modified RPE gDNA using the Repli-G Screening Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions.

    Article Title: DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer
    Article Snippet: .. In addition, whole genome amplified (WGA) DNA was constructed unsing the RepliG screening kit (Qiagen) as an absolute unmethylated DNA standard. ..

    Whole Genome Amplification:

    Article Title: t(3;17)(p25;q21) APL Represents a Cryptic Insertion of PML-RARA into the 3p25 Locus
    Article Snippet: .. To obtain adequate amounts of DNA for further analysis whole genome amplification was performed using REPLI-g kit (Qiagen). ..

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN). .. Mouse Htr3a expression levels in brain DMSTR were quantified by real-time PCR.

    Article Title: PIK3CA hotspot mutations in circulating tumor cells and paired circulating tumor DNA in breast cancer: a direct comparison study
    Article Snippet: Paragraph title: Whole genome amplification ... Amplification with REPLI‐g kit (Qiagen) was performed according to the manufacturer's recommendations.

    Article Title: Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements
    Article Snippet: .. One aliquot of each digested genomic DNA (20ng) was subjected to whole genome amplification respectively using REPLI-G kit (Qiagen) with 8 hours incubation at 30C. .. The amplified DNA sample was then purified by QIAEX II kit (Qiagen) with slightly modified protocol (3 instead of 2 washes with PE buffer and finally eluted in water rather than EB buffer).

    Article Title: Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model
    Article Snippet: .. Whole genome amplified (WGA) genomic DNA, which is completely unmethylated, was created using the Repli-g kit (Qiagen, Valencia, CA) following the manufacturer's protocol and was used as a negative control for MSP assays. .. The resulting DNA was sodium bisulfite modified as previously described [ ].

    Article Title: Opioid Receptor Polymorphism A118G Associated with Clinical Severity in a Drug Overdose Population
    Article Snippet: .. Whole genome amplification was performed using the Qiagen REPLI-g kit from approximately 5 ng of initial genomic DNA per sample, with cycling and hybridization conditions set according to the manufacturer's instructions to produce approximately 1–2 μg amplified DNA per sample. .. The OPRM1 polymorphisms (rs1799971 and rs2075572) were genotyped by the Taqman SNP Genotyping assay (Applied Biosystems, Foster City, CA, USA) by a blinded study investigator.

    Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes
    Article Snippet: .. DNA Whole Genome Amplification (WGA) was carried out on bisulfite-modified RPE gDNA using the Repli-G Screening Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. .. PCR amplicons were designed using PyroMark Assay Design SW 2.0 software (Qiagen, Hilden, Germany) (Additional file : Table S4) with a methylation standard curve used ensure the assay was without methylation bias [ ].

    Article Title: DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer
    Article Snippet: .. In addition, whole genome amplified (WGA) DNA was constructed unsing the RepliG screening kit (Qiagen) as an absolute unmethylated DNA standard. ..

    Article Title: Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth
    Article Snippet: .. Lysis, neutralization, and WGA were performed with the REPLI-g kit (Qiagen, Valencia, CA), using the recommended protocol except for on-chip WGA, for which the reaction mix was supplemented by 0.2% Tween 20 and one additional volume of polymerase. ..

    Article Title: Primary ciliary dyskinesia in Amish communities
    Article Snippet: .. Whole genome amplification (REPLI-g kit, Qiagen) was performed on each sample to provide adequate genetic material for testing. .. Focused, mutational analysis and sequencing of DNAI1 was performed.

    Positive Control:

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: To monitor both bisulfite conversion efficiency and accuracy of methylation detection, the CpG methylated NIH 3T3 mouse genomic DNA sample (New England Biolabs, Ipswich, MA) was taken as a positive control. .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN).

    Neutralization:

    Article Title: Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth
    Article Snippet: .. Lysis, neutralization, and WGA were performed with the REPLI-g kit (Qiagen, Valencia, CA), using the recommended protocol except for on-chip WGA, for which the reaction mix was supplemented by 0.2% Tween 20 and one additional volume of polymerase. ..

    Picogreen Assay:

    Article Title: PIK3CA hotspot mutations in circulating tumor cells and paired circulating tumor DNA in breast cancer: a direct comparison study
    Article Snippet: Amplification with REPLI‐g kit (Qiagen) was performed according to the manufacturer's recommendations. .. DNA concentration was determined using the Quant‐iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen™ , Waltham, MA, USA), and DNA was diluted according to the manufacturers’ manual.

    Construct:

    Article Title: DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer
    Article Snippet: .. In addition, whole genome amplified (WGA) DNA was constructed unsing the RepliG screening kit (Qiagen) as an absolute unmethylated DNA standard. ..

    Electrophoresis:

    Article Title: t(3;17)(p25;q21) APL Represents a Cryptic Insertion of PML-RARA into the 3p25 Locus
    Article Snippet: To obtain adequate amounts of DNA for further analysis whole genome amplification was performed using REPLI-g kit (Qiagen). .. Twenty μg of genomic DNA was digested with restriction enzyme KpnI (NEB) and fractionated by electrophoresis on a 1% agarose gel.

    Incubation:

    Article Title: Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements
    Article Snippet: .. One aliquot of each digested genomic DNA (20ng) was subjected to whole genome amplification respectively using REPLI-G kit (Qiagen) with 8 hours incubation at 30C. .. The amplified DNA sample was then purified by QIAEX II kit (Qiagen) with slightly modified protocol (3 instead of 2 washes with PE buffer and finally eluted in water rather than EB buffer).

    Formalin-fixed Paraffin-Embedded:

    Article Title: Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model
    Article Snippet: Genomic DNA was isolated either from frozen tissue samples using the Puregene kit (Qiagen, Valencia, CA) or from formalin fixed, paraffin embedded tissue sections using InstaGene Matrix (Bio-Rad, Hercules, CA) as previously published [ ]. .. Whole genome amplified (WGA) genomic DNA, which is completely unmethylated, was created using the Repli-g kit (Qiagen, Valencia, CA) following the manufacturer's protocol and was used as a negative control for MSP assays.

    Touchdown PCR:

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: A touchdown PCR using the FastStart Taq DNA Polymerase (Roche, Mannheim, Germany) was performed, including 3 cycles of 95°C 30 s/66°C 15 s/72°C 1 min, 3 cycles of 95°C 30 s/64°C 15 s/72°C 1 min, 3 cycles of 95°C 30 s/62°C 15 s/72°C 1 min, and 37 cycles of 95°C 30 s/60°C 15 s/72° C 1 min. After treatment with alkaline phosphatase ExoSAP-IT (Affymetrix, Santa Clara, CA), the PCR products were transcribed, cleaved by RNase A at specific bases (U or C), and spotted on a 384-pad SpectroCHIP (Sequenom) followed by spectral acquisition on a MassARRAY Analyzer. .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN).

    Modification:

    Article Title: Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements
    Article Snippet: One aliquot of each digested genomic DNA (20ng) was subjected to whole genome amplification respectively using REPLI-G kit (Qiagen) with 8 hours incubation at 30C. .. The amplified DNA sample was then purified by QIAEX II kit (Qiagen) with slightly modified protocol (3 instead of 2 washes with PE buffer and finally eluted in water rather than EB buffer).

    Article Title: Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model
    Article Snippet: Whole genome amplified (WGA) genomic DNA, which is completely unmethylated, was created using the Repli-g kit (Qiagen, Valencia, CA) following the manufacturer's protocol and was used as a negative control for MSP assays. .. The resulting DNA was sodium bisulfite modified as previously described [ ].

    Derivative Assay:

    Article Title: Primary ciliary dyskinesia in Amish communities
    Article Snippet: Whole genome amplification (REPLI-g kit, Qiagen) was performed on each sample to provide adequate genetic material for testing. .. Allele image profiles were generated using genotyping derived from 10K single nucleotide polymorphism arrays (Affimetrix) and microsatellite markers, short DNA segments with repeated, specific motifs 1–6 bases long, for candidate genes ( DNAI1, DNAH5, DNAH11, DNAH7, DNAH9, DNAI2, DNAL4, DNAH3 , and TCTEL1 ).

    Hybridization:

    Article Title: Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements
    Article Snippet: One aliquot of each digested genomic DNA (20ng) was subjected to whole genome amplification respectively using REPLI-G kit (Qiagen) with 8 hours incubation at 30C. .. 4 µg of the purified genomic DNA sample was submitted to Nimblegen for labeling and hybridization.

    Article Title: Opioid Receptor Polymorphism A118G Associated with Clinical Severity in a Drug Overdose Population
    Article Snippet: .. Whole genome amplification was performed using the Qiagen REPLI-g kit from approximately 5 ng of initial genomic DNA per sample, with cycling and hybridization conditions set according to the manufacturer's instructions to produce approximately 1–2 μg amplified DNA per sample. .. The OPRM1 polymorphisms (rs1799971 and rs2075572) were genotyped by the Taqman SNP Genotyping assay (Applied Biosystems, Foster City, CA, USA) by a blinded study investigator.

    Southern Blot:

    Article Title: t(3;17)(p25;q21) APL Represents a Cryptic Insertion of PML-RARA into the 3p25 Locus
    Article Snippet: Paragraph title: Southern Blot Analysis ... To obtain adequate amounts of DNA for further analysis whole genome amplification was performed using REPLI-g kit (Qiagen).

    Generated:

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN). .. Mouse Htr3a expression levels in brain DMSTR were quantified by real-time PCR.

    Article Title: Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model
    Article Snippet: Enzymatically methylated DNA was generated using SssI methylase (New England Biolabs, Beverly, MA) and used as a positive methylated DNA control for MSP assays. .. Whole genome amplified (WGA) genomic DNA, which is completely unmethylated, was created using the Repli-g kit (Qiagen, Valencia, CA) following the manufacturer's protocol and was used as a negative control for MSP assays.

    Article Title: Primary ciliary dyskinesia in Amish communities
    Article Snippet: Whole genome amplification (REPLI-g kit, Qiagen) was performed on each sample to provide adequate genetic material for testing. .. Allele image profiles were generated using genotyping derived from 10K single nucleotide polymorphism arrays (Affimetrix) and microsatellite markers, short DNA segments with repeated, specific motifs 1–6 bases long, for candidate genes ( DNAI1, DNAH5, DNAH11, DNAH7, DNAH9, DNAI2, DNAL4, DNAH3 , and TCTEL1 ).

    Negative Control:

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN). .. Mouse Htr3a expression levels in brain DMSTR were quantified by real-time PCR.

    Article Title: Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model
    Article Snippet: .. Whole genome amplified (WGA) genomic DNA, which is completely unmethylated, was created using the Repli-g kit (Qiagen, Valencia, CA) following the manufacturer's protocol and was used as a negative control for MSP assays. .. The resulting DNA was sodium bisulfite modified as previously described [ ].

    Polymerase Chain Reaction:

    Article Title: FUS GENE MUTATIONS IN FAMILIAL AND SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
    Article Snippet: - The FALS DNA samples were subsequently whole genome amplified in triplicate using the REPLI-g kit (Qiagen, Valencia, CA, USA). .. For all FALS and SALS patients, PCR of FUS exons 5, 6, 14 and 15 was performed with Qiagen products using primers located in adjacent introns.

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: A touchdown PCR using the FastStart Taq DNA Polymerase (Roche, Mannheim, Germany) was performed, including 3 cycles of 95°C 30 s/66°C 15 s/72°C 1 min, 3 cycles of 95°C 30 s/64°C 15 s/72°C 1 min, 3 cycles of 95°C 30 s/62°C 15 s/72°C 1 min, and 37 cycles of 95°C 30 s/60°C 15 s/72° C 1 min. After treatment with alkaline phosphatase ExoSAP-IT (Affymetrix, Santa Clara, CA), the PCR products were transcribed, cleaved by RNase A at specific bases (U or C), and spotted on a 384-pad SpectroCHIP (Sequenom) followed by spectral acquisition on a MassARRAY Analyzer. .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN).

    Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes
    Article Snippet: DNA Whole Genome Amplification (WGA) was carried out on bisulfite-modified RPE gDNA using the Repli-G Screening Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. .. PCR amplicons were designed using PyroMark Assay Design SW 2.0 software (Qiagen, Hilden, Germany) (Additional file : Table S4) with a methylation standard curve used ensure the assay was without methylation bias [ ].

    Article Title: DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer
    Article Snippet: Paragraph title: Quantitative Methylation Specific PCR (qMSP) ... In addition, whole genome amplified (WGA) DNA was constructed unsing the RepliG screening kit (Qiagen) as an absolute unmethylated DNA standard.

    Binding Assay:

    Article Title: DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer
    Article Snippet: During assay development, it became apparent that probes bearing minor groove binding moiety (Taqman MGB probes) provided a significant improvement in the assay specificity and, due to their smaller size, allowed for a more flexible assay design. .. In addition, whole genome amplified (WGA) DNA was constructed unsing the RepliG screening kit (Qiagen) as an absolute unmethylated DNA standard.

    Pyromark Assay:

    Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes
    Article Snippet: Assays were designed using PyroMark Assay Design Software (Version 2.0.1) to interrogate regions containing or surrounding ( < 100 bp upstream/downstream) candidate DML identified in our Illumina Human Methylation 450k BeadChip. .. DNA Whole Genome Amplification (WGA) was carried out on bisulfite-modified RPE gDNA using the Repli-G Screening Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions.

    DNA Extraction:

    Article Title: Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model
    Article Snippet: Paragraph title: DNA extraction and sodium bisulfite treatment ... Whole genome amplified (WGA) genomic DNA, which is completely unmethylated, was created using the Repli-g kit (Qiagen, Valencia, CA) following the manufacturer's protocol and was used as a negative control for MSP assays.

    Article Title: Primary ciliary dyskinesia in Amish communities
    Article Snippet: Blood or buccal cell samples were collected for genetic analysis; DNA was extracted using a commercially available DNA extraction kit. .. Whole genome amplification (REPLI-g kit, Qiagen) was performed on each sample to provide adequate genetic material for testing.

    Fluorescence:

    Article Title: Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements
    Article Snippet: Subsequent quantitation was done using PicoGreen fluorescence. .. One aliquot of each digested genomic DNA (20ng) was subjected to whole genome amplification respectively using REPLI-G kit (Qiagen) with 8 hours incubation at 30C.

    Methylation:

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN). .. Mouse Htr3a expression levels in brain DMSTR were quantified by real-time PCR.

    Article Title: Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model
    Article Snippet: Enzymatically methylated DNA was generated using SssI methylase (New England Biolabs, Beverly, MA) and used as a positive methylated DNA control for MSP assays. .. Whole genome amplified (WGA) genomic DNA, which is completely unmethylated, was created using the Repli-g kit (Qiagen, Valencia, CA) following the manufacturer's protocol and was used as a negative control for MSP assays.

    Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes
    Article Snippet: Paragraph title: Bisulfite pyrosequencing of candidate differentially methylated positions ... DNA Whole Genome Amplification (WGA) was carried out on bisulfite-modified RPE gDNA using the Repli-G Screening Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions.

    Article Title: DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer
    Article Snippet: Paragraph title: Quantitative Methylation Specific PCR (qMSP) ... In addition, whole genome amplified (WGA) DNA was constructed unsing the RepliG screening kit (Qiagen) as an absolute unmethylated DNA standard.

    Multiple Displacement Amplification:

    Article Title: Filtering "genic" open reading frames from genomic DNA samples for advanced annotation
    Article Snippet: .. DNA was amplified by multiple displacement amplification (MDA) with a Repli-g screening kit (Qiagen) according to the manufacturer’s instructions. .. After 16 h amplification, DNA was fragmented using nitrogen gas based nebulisation for 1 min at 45 psi; average fragment size was around 500 bp (200-800 bp range).

    Isolation:

    Article Title: Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model
    Article Snippet: Genomic DNA was isolated either from frozen tissue samples using the Puregene kit (Qiagen, Valencia, CA) or from formalin fixed, paraffin embedded tissue sections using InstaGene Matrix (Bio-Rad, Hercules, CA) as previously published [ ]. .. Whole genome amplified (WGA) genomic DNA, which is completely unmethylated, was created using the Repli-g kit (Qiagen, Valencia, CA) following the manufacturer's protocol and was used as a negative control for MSP assays.

    Size-exclusion Chromatography:

    Article Title: Filtering "genic" open reading frames from genomic DNA samples for advanced annotation
    Article Snippet: DNA was amplified by multiple displacement amplification (MDA) with a Repli-g screening kit (Qiagen) according to the manufacturer’s instructions. .. These vectors were obtained from the original pPAO phagemid vector [ ], by removing the g3p gene and either maintaining the Sec secretion leader (in POS vector) or replacing it with SRP and TAT leaders (in SOS and TOS respectively) encoded by oligonucleotides.

    Article Title: DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer
    Article Snippet: The reactions were performed on a 7500 FAST real time cycler (Applied Biosystems) under the following thermal profile: 95°C for 10 min, 50 cycles consisted of 95°C for 15 sec, 60°C-62°C for 1 min. .. In addition, whole genome amplified (WGA) DNA was constructed unsing the RepliG screening kit (Qiagen) as an absolute unmethylated DNA standard.

    Labeling:

    Article Title: Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements
    Article Snippet: One aliquot of each digested genomic DNA (20ng) was subjected to whole genome amplification respectively using REPLI-G kit (Qiagen) with 8 hours incubation at 30C. .. 4 µg of the purified genomic DNA sample was submitted to Nimblegen for labeling and hybridization.

    Purification:

    Article Title: Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements
    Article Snippet: One aliquot of each digested genomic DNA (20ng) was subjected to whole genome amplification respectively using REPLI-G kit (Qiagen) with 8 hours incubation at 30C. .. The amplified DNA sample was then purified by QIAEX II kit (Qiagen) with slightly modified protocol (3 instead of 2 washes with PE buffer and finally eluted in water rather than EB buffer).

    Article Title: Opioid Receptor Polymorphism A118G Associated with Clinical Severity in a Drug Overdose Population
    Article Snippet: DNA was extracted and purified in batches using the Qiagen QIAamp DNA blood mini kit. .. Whole genome amplification was performed using the Qiagen REPLI-g kit from approximately 5 ng of initial genomic DNA per sample, with cycling and hybridization conditions set according to the manufacturer's instructions to produce approximately 1–2 μg amplified DNA per sample.

    Sequencing:

    Article Title: FUS GENE MUTATIONS IN FAMILIAL AND SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
    Article Snippet: Due to the limited quantity and quality of DNA available in some of the samples, sequencing was restricted to those exons in which pathogenic FUS mutations had previously been reported. .. - The FALS DNA samples were subsequently whole genome amplified in triplicate using the REPLI-g kit (Qiagen, Valencia, CA, USA).

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: The reverse primer was tagged with the T7-promoter sequence for in vitro transcription. .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN).

    Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes
    Article Snippet: The percent methylation for each CpG site within the target sequence was calculated using the PyroQCpG Software (Qiagen) with non-CpG cytosine residues used as built-in controls to verify complete bisulfite conversion. .. DNA Whole Genome Amplification (WGA) was carried out on bisulfite-modified RPE gDNA using the Repli-G Screening Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions.

    Article Title: Primary ciliary dyskinesia in Amish communities
    Article Snippet: Whole genome amplification (REPLI-g kit, Qiagen) was performed on each sample to provide adequate genetic material for testing. .. Focused, mutational analysis and sequencing of DNAI1 was performed.

    Gel Extraction:

    Article Title: Filtering "genic" open reading frames from genomic DNA samples for advanced annotation
    Article Snippet: DNA was amplified by multiple displacement amplification (MDA) with a Repli-g screening kit (Qiagen) according to the manufacturer’s instructions. .. Fragments were twice blunt-ended (Quick blunt kit, NEB) and gel-purified (Gel extraction kit, Qiagen) before cloning into the EcoRV cleaved POS, SOS, and TOS vectors.

    Plasmid Preparation:

    Article Title: Filtering "genic" open reading frames from genomic DNA samples for advanced annotation
    Article Snippet: DNA was amplified by multiple displacement amplification (MDA) with a Repli-g screening kit (Qiagen) according to the manufacturer’s instructions. .. These vectors were obtained from the original pPAO phagemid vector [ ], by removing the g3p gene and either maintaining the Sec secretion leader (in POS vector) or replacing it with SRP and TAT leaders (in SOS and TOS respectively) encoded by oligonucleotides.

    Software:

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: The methylation calls were performed by the EpiTyper software v1.0 (Sequenom), which generates quantitative results (methyl CpG/total CpG) for each CpG site. .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN).

    Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes
    Article Snippet: The percent methylation for each CpG site within the target sequence was calculated using the PyroQCpG Software (Qiagen) with non-CpG cytosine residues used as built-in controls to verify complete bisulfite conversion. .. DNA Whole Genome Amplification (WGA) was carried out on bisulfite-modified RPE gDNA using the Repli-G Screening Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions.

    Functional Assay:

    Article Title: Opioid Receptor Polymorphism A118G Associated with Clinical Severity in a Drug Overdose Population
    Article Snippet: A118G (rs1799971) was chosen based on prior literature demonstrating associations with opioid tolerance [ , ] and the response to the opioid antidote naloxone [ , ]; a second SNP alternative splicing variant (rs2075572) was chosen based on prior literature implicating splicing variants with abuse behaviors [ ] and based on unpublished data from our own laboratory implicating functional opioid signaling abnormalities in the striatum. .. Whole genome amplification was performed using the Qiagen REPLI-g kit from approximately 5 ng of initial genomic DNA per sample, with cycling and hybridization conditions set according to the manufacturer's instructions to produce approximately 1–2 μg amplified DNA per sample.

    Variant Assay:

    Article Title: Opioid Receptor Polymorphism A118G Associated with Clinical Severity in a Drug Overdose Population
    Article Snippet: A118G (rs1799971) was chosen based on prior literature demonstrating associations with opioid tolerance [ , ] and the response to the opioid antidote naloxone [ , ]; a second SNP alternative splicing variant (rs2075572) was chosen based on prior literature implicating splicing variants with abuse behaviors [ ] and based on unpublished data from our own laboratory implicating functional opioid signaling abnormalities in the striatum. .. Whole genome amplification was performed using the Qiagen REPLI-g kit from approximately 5 ng of initial genomic DNA per sample, with cycling and hybridization conditions set according to the manufacturer's instructions to produce approximately 1–2 μg amplified DNA per sample.

    Agarose Gel Electrophoresis:

    Article Title: t(3;17)(p25;q21) APL Represents a Cryptic Insertion of PML-RARA into the 3p25 Locus
    Article Snippet: To obtain adequate amounts of DNA for further analysis whole genome amplification was performed using REPLI-g kit (Qiagen). .. Twenty μg of genomic DNA was digested with restriction enzyme KpnI (NEB) and fractionated by electrophoresis on a 1% agarose gel.

    In Vitro:

    Article Title: Ethanol-Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain
    Article Snippet: The reverse primer was tagged with the T7-promoter sequence for in vitro transcription. .. The negative control DNA sample (unmethylated) was generated by whole genome amplification of the CpG methylated NIH 3T3 mouse genomic DNA sample to remove methyl groups at CpG sites using reagents in the REPLI-g Kit (QIAGEN).

    Quantitation Assay:

    Article Title: Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements
    Article Snippet: Subsequent quantitation was done using PicoGreen fluorescence. .. One aliquot of each digested genomic DNA (20ng) was subjected to whole genome amplification respectively using REPLI-G kit (Qiagen) with 8 hours incubation at 30C.

    Concentration Assay:

    Article Title: PIK3CA hotspot mutations in circulating tumor cells and paired circulating tumor DNA in breast cancer: a direct comparison study
    Article Snippet: Amplification with REPLI‐g kit (Qiagen) was performed according to the manufacturer's recommendations. .. DNA concentration was determined using the Quant‐iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen™ , Waltham, MA, USA), and DNA was diluted according to the manufacturers’ manual.

    Lysis:

    Article Title: Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth
    Article Snippet: .. Lysis, neutralization, and WGA were performed with the REPLI-g kit (Qiagen, Valencia, CA), using the recommended protocol except for on-chip WGA, for which the reaction mix was supplemented by 0.2% Tween 20 and one additional volume of polymerase. ..

    TaqMan SNP Genotyping Assay:

    Article Title: Opioid Receptor Polymorphism A118G Associated with Clinical Severity in a Drug Overdose Population
    Article Snippet: Whole genome amplification was performed using the Qiagen REPLI-g kit from approximately 5 ng of initial genomic DNA per sample, with cycling and hybridization conditions set according to the manufacturer's instructions to produce approximately 1–2 μg amplified DNA per sample. .. The OPRM1 polymorphisms (rs1799971 and rs2075572) were genotyped by the Taqman SNP Genotyping assay (Applied Biosystems, Foster City, CA, USA) by a blinded study investigator.

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    Qiagen repli g single cell kit
    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) <t>REPLI-g.</t>
    Repli G Single Cell Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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: 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.

    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: 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.

    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: 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.

    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 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).

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