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Characteristics of the sequences used to assemble the Bison priscus mitochondrial genome. (A) Size distribution of the 3,851 unique Illumina reads. (B) Coverage for each position of the <t>SGE2seq</t> sequence obtained by merging the sequences of Illumina reads and <t>PCR</t> fragments.
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1) Product Images from "Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave"

Article Title: Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave

Journal: PLoS ONE

doi: 10.1371/journal.pone.0128267

Characteristics of the sequences used to assemble the Bison priscus mitochondrial genome. (A) Size distribution of the 3,851 unique Illumina reads. (B) Coverage for each position of the SGE2seq sequence obtained by merging the sequences of Illumina reads and PCR fragments.
Figure Legend Snippet: Characteristics of the sequences used to assemble the Bison priscus mitochondrial genome. (A) Size distribution of the 3,851 unique Illumina reads. (B) Coverage for each position of the SGE2seq sequence obtained by merging the sequences of Illumina reads and PCR fragments.

Techniques Used: Sequencing, Polymerase Chain Reaction

2) Product Images from "Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro"

Article Title: Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky219

Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.
Figure Legend Snippet: Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.

Techniques Used: CRISPR, Expressing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Variant Assay, Real-time Polymerase Chain Reaction, Standard Deviation

3) Product Images from "Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro"

Article Title: Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky219

Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C ) Mapping of spacers acquired from the G-1T variant target protospacer plasmid to the pUC19 backbone (see Supplementary Figure S7 for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D ) Position-dependent acquisition frequency for targets with seed mutation plotted over the acquisition frequency for the G-1T PAM mutation target. A high correlation between spacer acquisition patterns of all tested target variants (see Supplementary Figure S7B for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.
Figure Legend Snippet: Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C ) Mapping of spacers acquired from the G-1T variant target protospacer plasmid to the pUC19 backbone (see Supplementary Figure S7 for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D ) Position-dependent acquisition frequency for targets with seed mutation plotted over the acquisition frequency for the G-1T PAM mutation target. A high correlation between spacer acquisition patterns of all tested target variants (see Supplementary Figure S7B for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.

Techniques Used: CRISPR, Expressing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Variant Assay, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction, Standard Deviation

4) Product Images from "Novel endogenous simian retroviral integrations in Vero cells: implications for quality control of a human vaccine cell substrate"

Article Title: Novel endogenous simian retroviral integrations in Vero cells: implications for quality control of a human vaccine cell substrate

Journal: Scientific Reports

doi: 10.1038/s41598-017-18934-2

Structural comparison of SERV from different Vero sublines and different SVL. (a) Validation of Vero cell-specific SVL by genomic PCR. Nested PCR experiments were performed using genomic DNA from the three Vero cell sublines and AGM PBMC as described in the Methods. A schematic picture of nested PCR with the primer-binding sites is shown at the top. SVL IDs are specified under the panel. The black arrowheads indicate SERV-integrated PCR fragments, and the white arrowheads SERV-unintegrated PCR fragments. The arrow indicates an unknown band. Note that the sizes of smaller bands are consistent with the sizes expected for SERV-unintegrated genome sequences. 1st rd: 1st round PCR, 2nd rd: 2nd round PCR. All gel images shown include full-length without grouping. (b) A comparative analysis among twelve Vero cell-specific SVL regions. The colored bars show that the homologous region had at least 80% identity between the consensus SERV sequence found in the Vero 0111 genome (GenBank: AB935214) and each SERV region. Genetic features including mutations for respective SVLs are shown on the right side. Identity with 5′-LTR and 3′-LTR sequences of the AB935214 reference in the BLASTN homology search are: 94.3 and 73.4% for SVL13c, 72.0 and 99.0% for SVL15f, 95.4 and 74.0% for SVL22e, and 94.7 and 74.2% for SVL26, respectively. Due to the high identities with both LTRs of the reference, both 5′- and 3′-LTR regions of these SVL integrations are depicted in the panel, although the SVLs with no intervening sequence (13c, 15f, 22e, and 26) are the solo LTRs as highlighted on the right side of the panel.
Figure Legend Snippet: Structural comparison of SERV from different Vero sublines and different SVL. (a) Validation of Vero cell-specific SVL by genomic PCR. Nested PCR experiments were performed using genomic DNA from the three Vero cell sublines and AGM PBMC as described in the Methods. A schematic picture of nested PCR with the primer-binding sites is shown at the top. SVL IDs are specified under the panel. The black arrowheads indicate SERV-integrated PCR fragments, and the white arrowheads SERV-unintegrated PCR fragments. The arrow indicates an unknown band. Note that the sizes of smaller bands are consistent with the sizes expected for SERV-unintegrated genome sequences. 1st rd: 1st round PCR, 2nd rd: 2nd round PCR. All gel images shown include full-length without grouping. (b) A comparative analysis among twelve Vero cell-specific SVL regions. The colored bars show that the homologous region had at least 80% identity between the consensus SERV sequence found in the Vero 0111 genome (GenBank: AB935214) and each SERV region. Genetic features including mutations for respective SVLs are shown on the right side. Identity with 5′-LTR and 3′-LTR sequences of the AB935214 reference in the BLASTN homology search are: 94.3 and 73.4% for SVL13c, 72.0 and 99.0% for SVL15f, 95.4 and 74.0% for SVL22e, and 94.7 and 74.2% for SVL26, respectively. Due to the high identities with both LTRs of the reference, both 5′- and 3′-LTR regions of these SVL integrations are depicted in the panel, although the SVLs with no intervening sequence (13c, 15f, 22e, and 26) are the solo LTRs as highlighted on the right side of the panel.

Techniques Used: Polymerase Chain Reaction, Nested PCR, Binding Assay, Sequencing

5) Product Images from "Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex"

Article Title: Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1602639113

CRISPR interference and primed CRISPR adaptation against matching and partially matching protospacer targets located on plasmids. ( A ) The E. coli KD263 cells capable of inducible cas gene expression and carrying an engineered CRISPR array with a single g8 spacer are schematically shown. E. coli KD263 was transformed with ampicillin-resistant pT7Blue vector ( B ) or pT7Blue-based plasmid pG8 ( C ) with a fully matching g8 protospacer with a functional PAM or pG8mut ( D ), carrying, respectively, a protospacer with a functional PAM and a C1T mutation in the protospacer. Protospacers are shown as blue arrows with the arrowpoint directed away from the 5′-ATG-3′ PAM. The C1T mutation is shown as a yellow star. Graphs show the number of ampicillin-resistant CFUs in cultures transformed with each plasmid with or without induction of cas gene expression. Plasmid DNA was purified from cells collected at the times indicated and resolved by agarose gel electrophoresis. The products of PCR amplification with primers annealing at the g8 spacer and further upstream in the CRISPR array leader are shown at the bottom.
Figure Legend Snippet: CRISPR interference and primed CRISPR adaptation against matching and partially matching protospacer targets located on plasmids. ( A ) The E. coli KD263 cells capable of inducible cas gene expression and carrying an engineered CRISPR array with a single g8 spacer are schematically shown. E. coli KD263 was transformed with ampicillin-resistant pT7Blue vector ( B ) or pT7Blue-based plasmid pG8 ( C ) with a fully matching g8 protospacer with a functional PAM or pG8mut ( D ), carrying, respectively, a protospacer with a functional PAM and a C1T mutation in the protospacer. Protospacers are shown as blue arrows with the arrowpoint directed away from the 5′-ATG-3′ PAM. The C1T mutation is shown as a yellow star. Graphs show the number of ampicillin-resistant CFUs in cultures transformed with each plasmid with or without induction of cas gene expression. Plasmid DNA was purified from cells collected at the times indicated and resolved by agarose gel electrophoresis. The products of PCR amplification with primers annealing at the g8 spacer and further upstream in the CRISPR array leader are shown at the bottom.

Techniques Used: CRISPR, Expressing, Transformation Assay, Plasmid Preparation, Functional Assay, Mutagenesis, Purification, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification

CRISPR adaptation from matching and partially matching protospacer targets located in an in cis configuration. ( A ) The E. coli KD263 cells transformed with pT7Blue-based RwFm or RmFw plasmids carrying a wild-type and a mutant, C1T, version of the g8 protospacer on different strands are schematically shown at the top. ( B ) A graph showing the number of CFUs on LB plates containing ampicillin in KD263 cultures transformed with one of the plasmids shown in A with or without induction of cas gene expression. Shown below are the results of agarose gel electrophoresis of PCR amplification products obtained from aliquots of induced culture with primers annealing at the g8 spacer and further upstream in the KD263 CRISPR array leader. ( C ) Bar graphs showing the overall statistics of acquired spacers in each cell culture shown in A with spacers mapped to either plasmid DNA strand shown in different colors (color coded as in plasmid schematics in A ). ( D ) Mapping of spacers acquired by KD263 cells transformed with RwFm or RmFw. The positions of priming protospacers are shown by blue arrows.
Figure Legend Snippet: CRISPR adaptation from matching and partially matching protospacer targets located in an in cis configuration. ( A ) The E. coli KD263 cells transformed with pT7Blue-based RwFm or RmFw plasmids carrying a wild-type and a mutant, C1T, version of the g8 protospacer on different strands are schematically shown at the top. ( B ) A graph showing the number of CFUs on LB plates containing ampicillin in KD263 cultures transformed with one of the plasmids shown in A with or without induction of cas gene expression. Shown below are the results of agarose gel electrophoresis of PCR amplification products obtained from aliquots of induced culture with primers annealing at the g8 spacer and further upstream in the KD263 CRISPR array leader. ( C ) Bar graphs showing the overall statistics of acquired spacers in each cell culture shown in A with spacers mapped to either plasmid DNA strand shown in different colors (color coded as in plasmid schematics in A ). ( D ) Mapping of spacers acquired by KD263 cells transformed with RwFm or RmFw. The positions of priming protospacers are shown by blue arrows.

Techniques Used: CRISPR, Transformation Assay, Mutagenesis, Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Cell Culture, Plasmid Preparation

Increased expression of cas1 and cas2 leads to primed adaptation from fully matching protospacer targets. ( A ) An agarose gel showing the results of separation of products of PCR amplification of CRISPR arrays from aliquots of KD263 (lanes 1 and 2) and KD546 (lanes 3 and 4) transformed with indicated plasmids. ( B ) on donor plasmids.
Figure Legend Snippet: Increased expression of cas1 and cas2 leads to primed adaptation from fully matching protospacer targets. ( A ) An agarose gel showing the results of separation of products of PCR amplification of CRISPR arrays from aliquots of KD263 (lanes 1 and 2) and KD546 (lanes 3 and 4) transformed with indicated plasmids. ( B ) on donor plasmids.

Techniques Used: Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, CRISPR, Transformation Assay

6) Product Images from "High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli"

Article Title: High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli

Journal: RNA Biology

doi: 10.4161/rna.24325

Figure 1. Experimental set-up to monitor CRISPR spacer acquisition. ( A ) At the top, the E. coli BL21 AI CRISPR 2.3 cassette is schematically presented, with repeats indicated as numbered gray rectangles. A leftward arrow indicates CRISPR promoter located in the leader sequence. The primers used to amplify the leader-proximal end of the cassette are shown (thick, not annealed parts of primers correspond to barcodes for high-throughput sequencing). Below, the structures of amplified DNA fragments expected in the absence (middle) or in the presence (bottom) of spacer acquisition are shown. ( B ) Results of PCR amplification using the primer set shown in ( A ) of DNA prepared from E. coli BL21 AI cultures transformed with plasmids pT7blue (lanes 1 and 3) or P1 and P2 plasmids (lanes 2 and 4) after an overnight growth at conditions of induction of plasmid-borne CRISPR/Cas components and in the absence of ampicillin needed to maintain pT7blue and its derivatives. The gray arrow indicates a PCR fragment arising from amplification of leader-proximal end of unexpanded CRSIRP 2.3 cassette; black arrow indicates a PCR fragment arising from amplification of CRISPR 2.3 cassette expanded by one spacer-repeat unit. Numbers at the left-hand side of the gel indicate the lengths (in bp) of DNA size markers. ( C ) Statistics of high-throughput sequencing of PCR amplification products extended by one spacer-repeat unit obtained with P1 and P2 samples.
Figure Legend Snippet: Figure 1. Experimental set-up to monitor CRISPR spacer acquisition. ( A ) At the top, the E. coli BL21 AI CRISPR 2.3 cassette is schematically presented, with repeats indicated as numbered gray rectangles. A leftward arrow indicates CRISPR promoter located in the leader sequence. The primers used to amplify the leader-proximal end of the cassette are shown (thick, not annealed parts of primers correspond to barcodes for high-throughput sequencing). Below, the structures of amplified DNA fragments expected in the absence (middle) or in the presence (bottom) of spacer acquisition are shown. ( B ) Results of PCR amplification using the primer set shown in ( A ) of DNA prepared from E. coli BL21 AI cultures transformed with plasmids pT7blue (lanes 1 and 3) or P1 and P2 plasmids (lanes 2 and 4) after an overnight growth at conditions of induction of plasmid-borne CRISPR/Cas components and in the absence of ampicillin needed to maintain pT7blue and its derivatives. The gray arrow indicates a PCR fragment arising from amplification of leader-proximal end of unexpanded CRSIRP 2.3 cassette; black arrow indicates a PCR fragment arising from amplification of CRISPR 2.3 cassette expanded by one spacer-repeat unit. Numbers at the left-hand side of the gel indicate the lengths (in bp) of DNA size markers. ( C ) Statistics of high-throughput sequencing of PCR amplification products extended by one spacer-repeat unit obtained with P1 and P2 samples.

Techniques Used: CRISPR, Sequencing, Next-Generation Sequencing, Amplification, Polymerase Chain Reaction, Transformation Assay, Plasmid Preparation

7) Product Images from "Mutant TRP53 exerts a target gene-selective dominant-negative effect to drive tumor development"

Article Title: Mutant TRP53 exerts a target gene-selective dominant-negative effect to drive tumor development

Journal: Genes & Development

doi: 10.1101/gad.314286.118

Overexpression of mutant TRP53 proteins accelerates lymphoma development in an Eμ-Myc;Trp53 +/+ background and relieves selective pressure for mutation of endogenous Trp53 genes. ( A ) Kaplan-Meier survival curves for mice reconstituted with Eμ-Myc;Trp53 +/+ HSPCs comparing empty vector control (pMIG), CRISPR/Cas9 Trp53 knockout , and each mutant TRP53 protein (V170M, I192S, G280, R246Q, and R270H). P -values were determined by log rank (Mantel-Cox) test. ( B ) Selected TRP53 protein immunohistochemistry in lymphomas from the Eμ-Myc hematopoietic reconstitution experiments (mice #88 and #541 plus control mouse #53). ( C ) Endogenous Trp53 allele copy number in lymphomas from the Eμ-Myc hematopoietic reconstitution experiments as determined by genomic DNA quantitative PCR (pMIG/control: #53; V170M: #55, #66, and #541; G280: #68 and #81; I192S: #72 and #546; R246Q: #97 and #98; R270H: #80, #543, and #544). Primary cells from Trp53 −/− and Trp53 +/− mice were used as controls. Data from MiSeq analysis throughout the coding region of the DNA-binding domain (exons 4–10) are indicated. (wt) Wild-type sequence. Data represent mean ± SEM. (*) P
Figure Legend Snippet: Overexpression of mutant TRP53 proteins accelerates lymphoma development in an Eμ-Myc;Trp53 +/+ background and relieves selective pressure for mutation of endogenous Trp53 genes. ( A ) Kaplan-Meier survival curves for mice reconstituted with Eμ-Myc;Trp53 +/+ HSPCs comparing empty vector control (pMIG), CRISPR/Cas9 Trp53 knockout , and each mutant TRP53 protein (V170M, I192S, G280, R246Q, and R270H). P -values were determined by log rank (Mantel-Cox) test. ( B ) Selected TRP53 protein immunohistochemistry in lymphomas from the Eμ-Myc hematopoietic reconstitution experiments (mice #88 and #541 plus control mouse #53). ( C ) Endogenous Trp53 allele copy number in lymphomas from the Eμ-Myc hematopoietic reconstitution experiments as determined by genomic DNA quantitative PCR (pMIG/control: #53; V170M: #55, #66, and #541; G280: #68 and #81; I192S: #72 and #546; R246Q: #97 and #98; R270H: #80, #543, and #544). Primary cells from Trp53 −/− and Trp53 +/− mice were used as controls. Data from MiSeq analysis throughout the coding region of the DNA-binding domain (exons 4–10) are indicated. (wt) Wild-type sequence. Data represent mean ± SEM. (*) P

Techniques Used: Over Expression, Mutagenesis, Mouse Assay, Plasmid Preparation, CRISPR, Knock-Out, Immunohistochemistry, Real-time Polymerase Chain Reaction, Binding Assay, Sequencing

8) Product Images from "Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome"

Article Title: Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.119.400613

Schematic of the construction of Ta subfamily enriched L1Hs sequencing libraries. gDNA isolated from NeuN+ nuclei was enzymatically digested with HaeIII in the presence of shrimp alkaline phosphatase (rSAP) to fragment the genome and remove 5′ phosphates from cleavage products. A single primer extension using the Ta subfamily specific L1HsACA primer extends the 3′ end of the L1 sequence into the downstream gDNA. The 3′ ‘A’ overhang from the single primer extension is ligated to a custom T-linker, and primary PCR amplifies the construct using L1HsACA and T-linker specific primers. Hemi-nested secondary PCR using the L1Hs specific L1HsG primer and T-linker primer reduces the length of the L1 sequence carried forward and adds a sequencing adapter to the L1 end. Tertiary PCR uses primers complementary to the 5′ end of library amplicons to add a barcode to the L1 end and Illumina flow cell adapters to both ends of the amplicons.
Figure Legend Snippet: Schematic of the construction of Ta subfamily enriched L1Hs sequencing libraries. gDNA isolated from NeuN+ nuclei was enzymatically digested with HaeIII in the presence of shrimp alkaline phosphatase (rSAP) to fragment the genome and remove 5′ phosphates from cleavage products. A single primer extension using the Ta subfamily specific L1HsACA primer extends the 3′ end of the L1 sequence into the downstream gDNA. The 3′ ‘A’ overhang from the single primer extension is ligated to a custom T-linker, and primary PCR amplifies the construct using L1HsACA and T-linker specific primers. Hemi-nested secondary PCR using the L1Hs specific L1HsG primer and T-linker primer reduces the length of the L1 sequence carried forward and adds a sequencing adapter to the L1 end. Tertiary PCR uses primers complementary to the 5′ end of library amplicons to add a barcode to the L1 end and Illumina flow cell adapters to both ends of the amplicons.

Techniques Used: Sequencing, Isolation, Polymerase Chain Reaction, Construct

Related Articles

Polymerase Chain Reaction:

Article Title: Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex
Article Snippet: .. PCR fragments corresponding to expanded CRISPR arrays were subjected to Illumina sequencing, and extracted sequences of acquired spacers were mapped. .. For each plasmid pair analyzed, most (97% and 99%) spacers originated from a plasmid that carried the mutant protospacer, and a strong (81% and 89%) bias for selection of spacers from a nontargeted strand was observed ( , Top ).

Article Title: Mutant TRP53 exerts a target gene-selective dominant-negative effect to drive tumor development
Article Snippet: .. PCR fragments were sequenced using the MiSeq platform (Illumina). .. The inducible lentiviral platform for CRISPR/Cas9 gene modification using doxycycline-inducible sgRNA expression and stable constitutive expression of CAS9 and the sgRNA sequences targeting exon 4 and exon 5 of the mouse Trp53 gene have been described previously ( ).

Article Title: Novel endogenous simian retroviral integrations in Vero cells: implications for quality control of a human vaccine cell substrate
Article Snippet: .. Paired-end libraries of the PCR fragments from SVL integrations (SVL ID; 3b, 4e, 6a, 10b, 20d, 21b, 21e, and 27b) were prepared for MiniSeq sequencing using the Nextera XT DNA Library Preparation Kit (Illumina) and sequenced with the MiniSeq Mid Output Kit (Illumina). .. The Illumina short reads of long-PCR fragments were assembled using the A5-MiSeq program , followed by verification of the 5′ and 3′ end regions containing LTR regions using Sanger sequencing data.

Article Title: Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome
Article Snippet: .. The barcoded, L1-enriched sequencing libraries were quantified, in triplicate, for the concentration of PCR fragments properly flanked by the Illumina flow cell adapters using the KAPA Library Quantification Kit (Roche #KK4835) and 7900HT real-time PCR (Applied Biosystems) according to the manufacturer’s protocol, utilizing 1:20,000, 1:200,000 and 1:2,000,000 dilutions. .. Using the KAPA Library Quantification Analysis Template_ILM_v4-1, the Ct values and the average fragment size (determined above by Bioanalyzer), the concentration of sequenceable library was calculated for each sample.

Article Title: Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave
Article Snippet: .. The final sequence, termed SGE2seq, was established using 81 consensus sequences of PCR fragments to complement the information derived from the Illumina reads. .. All the Illumina reads were aligned one last time to SGE2seq, which yielded 3,851 unique reads.

Article Title: Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro
Article Snippet: .. For all target variants that supported priming, PCR fragments corresponding to expanded CRISPR arrays were subjected to Illumina sequencing. .. After filtering, the acquired spacer sequences were extracted and mapped onto the donor plasmid backbone (Figure , ).

Article Title: High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli
Article Snippet: .. The stimulation of CRISPR adaptation in CRISPR/Cas expressing cells harboring protospacer-containing plasmids is thus due to priming., PCR fragments corresponding to CRISPR 2.3 cassettes extended by one spacer-repeat unit were subjected to MySeq Illumina sequencing. .. Barcoding allowed simultaneous monitoring of sequences from cultures of cells that have lost P1 and P2 plasmids.

Real-time Polymerase Chain Reaction:

Article Title: Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome
Article Snippet: .. The barcoded, L1-enriched sequencing libraries were quantified, in triplicate, for the concentration of PCR fragments properly flanked by the Illumina flow cell adapters using the KAPA Library Quantification Kit (Roche #KK4835) and 7900HT real-time PCR (Applied Biosystems) according to the manufacturer’s protocol, utilizing 1:20,000, 1:200,000 and 1:2,000,000 dilutions. .. Using the KAPA Library Quantification Analysis Template_ILM_v4-1, the Ct values and the average fragment size (determined above by Bioanalyzer), the concentration of sequenceable library was calculated for each sample.

Concentration Assay:

Article Title: Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome
Article Snippet: .. The barcoded, L1-enriched sequencing libraries were quantified, in triplicate, for the concentration of PCR fragments properly flanked by the Illumina flow cell adapters using the KAPA Library Quantification Kit (Roche #KK4835) and 7900HT real-time PCR (Applied Biosystems) according to the manufacturer’s protocol, utilizing 1:20,000, 1:200,000 and 1:2,000,000 dilutions. .. Using the KAPA Library Quantification Analysis Template_ILM_v4-1, the Ct values and the average fragment size (determined above by Bioanalyzer), the concentration of sequenceable library was calculated for each sample.

Expressing:

Article Title: High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli
Article Snippet: .. The stimulation of CRISPR adaptation in CRISPR/Cas expressing cells harboring protospacer-containing plasmids is thus due to priming., PCR fragments corresponding to CRISPR 2.3 cassettes extended by one spacer-repeat unit were subjected to MySeq Illumina sequencing. .. Barcoding allowed simultaneous monitoring of sequences from cultures of cells that have lost P1 and P2 plasmids.

CRISPR:

Article Title: Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex
Article Snippet: .. PCR fragments corresponding to expanded CRISPR arrays were subjected to Illumina sequencing, and extracted sequences of acquired spacers were mapped. .. For each plasmid pair analyzed, most (97% and 99%) spacers originated from a plasmid that carried the mutant protospacer, and a strong (81% and 89%) bias for selection of spacers from a nontargeted strand was observed ( , Top ).

Article Title: Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro
Article Snippet: .. For all target variants that supported priming, PCR fragments corresponding to expanded CRISPR arrays were subjected to Illumina sequencing. .. After filtering, the acquired spacer sequences were extracted and mapped onto the donor plasmid backbone (Figure , ).

Article Title: High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli
Article Snippet: .. The stimulation of CRISPR adaptation in CRISPR/Cas expressing cells harboring protospacer-containing plasmids is thus due to priming., PCR fragments corresponding to CRISPR 2.3 cassettes extended by one spacer-repeat unit were subjected to MySeq Illumina sequencing. .. Barcoding allowed simultaneous monitoring of sequences from cultures of cells that have lost P1 and P2 plasmids.

Sequencing:

Article Title: Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex
Article Snippet: .. PCR fragments corresponding to expanded CRISPR arrays were subjected to Illumina sequencing, and extracted sequences of acquired spacers were mapped. .. For each plasmid pair analyzed, most (97% and 99%) spacers originated from a plasmid that carried the mutant protospacer, and a strong (81% and 89%) bias for selection of spacers from a nontargeted strand was observed ( , Top ).

Article Title: Novel endogenous simian retroviral integrations in Vero cells: implications for quality control of a human vaccine cell substrate
Article Snippet: .. Paired-end libraries of the PCR fragments from SVL integrations (SVL ID; 3b, 4e, 6a, 10b, 20d, 21b, 21e, and 27b) were prepared for MiniSeq sequencing using the Nextera XT DNA Library Preparation Kit (Illumina) and sequenced with the MiniSeq Mid Output Kit (Illumina). .. The Illumina short reads of long-PCR fragments were assembled using the A5-MiSeq program , followed by verification of the 5′ and 3′ end regions containing LTR regions using Sanger sequencing data.

Article Title: Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome
Article Snippet: .. The barcoded, L1-enriched sequencing libraries were quantified, in triplicate, for the concentration of PCR fragments properly flanked by the Illumina flow cell adapters using the KAPA Library Quantification Kit (Roche #KK4835) and 7900HT real-time PCR (Applied Biosystems) according to the manufacturer’s protocol, utilizing 1:20,000, 1:200,000 and 1:2,000,000 dilutions. .. Using the KAPA Library Quantification Analysis Template_ILM_v4-1, the Ct values and the average fragment size (determined above by Bioanalyzer), the concentration of sequenceable library was calculated for each sample.

Article Title: Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave
Article Snippet: .. The final sequence, termed SGE2seq, was established using 81 consensus sequences of PCR fragments to complement the information derived from the Illumina reads. .. All the Illumina reads were aligned one last time to SGE2seq, which yielded 3,851 unique reads.

Article Title: Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro
Article Snippet: .. For all target variants that supported priming, PCR fragments corresponding to expanded CRISPR arrays were subjected to Illumina sequencing. .. After filtering, the acquired spacer sequences were extracted and mapped onto the donor plasmid backbone (Figure , ).

Article Title: High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli
Article Snippet: .. The stimulation of CRISPR adaptation in CRISPR/Cas expressing cells harboring protospacer-containing plasmids is thus due to priming., PCR fragments corresponding to CRISPR 2.3 cassettes extended by one spacer-repeat unit were subjected to MySeq Illumina sequencing. .. Barcoding allowed simultaneous monitoring of sequences from cultures of cells that have lost P1 and P2 plasmids.

Derivative Assay:

Article Title: Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave
Article Snippet: .. The final sequence, termed SGE2seq, was established using 81 consensus sequences of PCR fragments to complement the information derived from the Illumina reads. .. All the Illumina reads were aligned one last time to SGE2seq, which yielded 3,851 unique reads.

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    Illumina Inc pcr fragments
    Characteristics of the sequences used to assemble the Bison priscus mitochondrial genome. (A) Size distribution of the 3,851 unique Illumina reads. (B) Coverage for each position of the <t>SGE2seq</t> sequence obtained by merging the sequences of Illumina reads and <t>PCR</t> fragments.
    Pcr Fragments, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Characteristics of the sequences used to assemble the Bison priscus mitochondrial genome. (A) Size distribution of the 3,851 unique Illumina reads. (B) Coverage for each position of the SGE2seq sequence obtained by merging the sequences of Illumina reads and PCR fragments.

    Journal: PLoS ONE

    Article Title: Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave

    doi: 10.1371/journal.pone.0128267

    Figure Lengend Snippet: Characteristics of the sequences used to assemble the Bison priscus mitochondrial genome. (A) Size distribution of the 3,851 unique Illumina reads. (B) Coverage for each position of the SGE2seq sequence obtained by merging the sequences of Illumina reads and PCR fragments.

    Article Snippet: The final sequence, termed SGE2seq, was established using 81 consensus sequences of PCR fragments to complement the information derived from the Illumina reads.

    Techniques: Sequencing, Polymerase Chain Reaction

    Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.

    Journal: Nucleic Acids Research

    Article Title: Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro

    doi: 10.1093/nar/gky219

    Figure Lengend Snippet: Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.

    Article Snippet: For all target variants that supported priming, PCR fragments corresponding to expanded CRISPR arrays were subjected to Illumina sequencing.

    Techniques: CRISPR, Expressing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Variant Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C ) Mapping of spacers acquired from the G-1T variant target protospacer plasmid to the pUC19 backbone (see Supplementary Figure S7 for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D ) Position-dependent acquisition frequency for targets with seed mutation plotted over the acquisition frequency for the G-1T PAM mutation target. A high correlation between spacer acquisition patterns of all tested target variants (see Supplementary Figure S7B for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.

    Journal: Nucleic Acids Research

    Article Title: Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro

    doi: 10.1093/nar/gky219

    Figure Lengend Snippet: Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C ) Mapping of spacers acquired from the G-1T variant target protospacer plasmid to the pUC19 backbone (see Supplementary Figure S7 for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D ) Position-dependent acquisition frequency for targets with seed mutation plotted over the acquisition frequency for the G-1T PAM mutation target. A high correlation between spacer acquisition patterns of all tested target variants (see Supplementary Figure S7B for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.

    Article Snippet: For all target variants that supported priming, PCR fragments corresponding to expanded CRISPR arrays were subjected to Illumina sequencing.

    Techniques: CRISPR, Expressing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Variant Assay, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction, Standard Deviation

    Effects of AthCV1 infection on the colony morphology of Aspergillus species. ( A ) polymerase chain reaction (PCR) assay showing successful AthCV1 transfection to different virus-free Aspergillus species; reverse transcription PCR, lane L = 1 kb plus DNA ladder; -Ve= PCR negative control (RNA sample was replaced with ultrapure water), +Ath1 = AthCV1 +ve control; +fu = AthCV1 transfected A. fumigatus (Afu-13); +ng = AthCV1 transfected A. niger (Ang-9); +nd = AthCV1 transfected A. nidulans (And-1); –fu, –ng and –nd = original virus-free isolates of A. fumigatus (Afu-13), A. niger (Ang-9) and A. nidulans (And-1), respectively. ( B ) Afu-13 grown at 37 °C; normal growth in the virus-free isolate, sectors formed in isogenic AthCV1-transfected line (arrow). ( C ) Afu-13 grown at 20 °C; normal growth in the virus-free isolate, sectors formed in isogenic AthCV1-transfected line (arrow). ( D ) And-1 grown at 37 °C; normal growth in the virus-free isolate, ascospore-rich sectors formed in isogenic AthCV1-transfected line (arrow). ( E ) And-1 grown at 37 °C; normal growth in both virus-free and AthCV1-transfected lines. ( F ) Ang-9 grown at 37 °C; normal growth in the virus-free isolate, conidial-free sectors with sclerotia formed (arrow) in AthCV1-transfected line. ( G ) Ang-9 grown at 20 °C; normal growth in the virus-free isolate, conidial- and sclerotia-free sectors in AthCV1-transfected line (arrow).

    Journal: Viruses

    Article Title: The Effect of Aspergillus Thermomutatus Chrysovirus 1 on the Biology of Three Aspergillus Species

    doi: 10.3390/v10100539

    Figure Lengend Snippet: Effects of AthCV1 infection on the colony morphology of Aspergillus species. ( A ) polymerase chain reaction (PCR) assay showing successful AthCV1 transfection to different virus-free Aspergillus species; reverse transcription PCR, lane L = 1 kb plus DNA ladder; -Ve= PCR negative control (RNA sample was replaced with ultrapure water), +Ath1 = AthCV1 +ve control; +fu = AthCV1 transfected A. fumigatus (Afu-13); +ng = AthCV1 transfected A. niger (Ang-9); +nd = AthCV1 transfected A. nidulans (And-1); –fu, –ng and –nd = original virus-free isolates of A. fumigatus (Afu-13), A. niger (Ang-9) and A. nidulans (And-1), respectively. ( B ) Afu-13 grown at 37 °C; normal growth in the virus-free isolate, sectors formed in isogenic AthCV1-transfected line (arrow). ( C ) Afu-13 grown at 20 °C; normal growth in the virus-free isolate, sectors formed in isogenic AthCV1-transfected line (arrow). ( D ) And-1 grown at 37 °C; normal growth in the virus-free isolate, ascospore-rich sectors formed in isogenic AthCV1-transfected line (arrow). ( E ) And-1 grown at 37 °C; normal growth in both virus-free and AthCV1-transfected lines. ( F ) Ang-9 grown at 37 °C; normal growth in the virus-free isolate, conidial-free sectors with sclerotia formed (arrow) in AthCV1-transfected line. ( G ) Ang-9 grown at 20 °C; normal growth in the virus-free isolate, conidial- and sclerotia-free sectors in AthCV1-transfected line (arrow).

    Article Snippet: An Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) with a DNA 1000 chip was used to verify the size of PCR-enriched fragments and for library quantification qPCR was used, according to the Illumina qPCR Quantification Protocol Guide (# 11322363) (Illumina, San Diego, CA, USA).

    Techniques: Infection, Polymerase Chain Reaction, Transfection, Negative Control