genome sequencing wgs  (Covaris)


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    Structured Review

    Covaris genome sequencing wgs
    Optimization of <t>Kmar</t> de novo genome assemblies. Raw reads for pair-ended Kmar <t>WGS</t> were assembled at various k-mer values to build a de novo genome. The graph shows a positive correlation between higher k-mer values and Kmar genome size. A similar correlation was found for N50, but peaked at k = 60. Thus, a Kmar genome with the highest N50 value (32,044 bp) that yielded a genome size of 642,279,823 bp was selected to serve as a reference genome for the RNA-Seq data analyses. [Note: k-mer at higher than k = 64 failed to assemble a de novo genome]
    Genome Sequencing Wgs, supplied by Covaris, used in various techniques. Bioz Stars score: 89/100, based on 809 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 89 stars, based on 809 article reviews
    Price from $9.99 to $1999.99
    genome sequencing wgs - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Transcriptomics of diapause in an isogenic self-fertilizing vertebrate"

    Article Title: Transcriptomics of diapause in an isogenic self-fertilizing vertebrate

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-2210-0

    Optimization of Kmar de novo genome assemblies. Raw reads for pair-ended Kmar WGS were assembled at various k-mer values to build a de novo genome. The graph shows a positive correlation between higher k-mer values and Kmar genome size. A similar correlation was found for N50, but peaked at k = 60. Thus, a Kmar genome with the highest N50 value (32,044 bp) that yielded a genome size of 642,279,823 bp was selected to serve as a reference genome for the RNA-Seq data analyses. [Note: k-mer at higher than k = 64 failed to assemble a de novo genome]
    Figure Legend Snippet: Optimization of Kmar de novo genome assemblies. Raw reads for pair-ended Kmar WGS were assembled at various k-mer values to build a de novo genome. The graph shows a positive correlation between higher k-mer values and Kmar genome size. A similar correlation was found for N50, but peaked at k = 60. Thus, a Kmar genome with the highest N50 value (32,044 bp) that yielded a genome size of 642,279,823 bp was selected to serve as a reference genome for the RNA-Seq data analyses. [Note: k-mer at higher than k = 64 failed to assemble a de novo genome]

    Techniques Used: RNA Sequencing Assay

    2) Product Images from "The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi"

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061778

    The rRNA/tRNA gene cluster of Plasmodium chabaudi chabaudi CB plastid DNA. (A) Part of the Pcc CB plastid DNA was amplified by PCR from total parasite DNA with the primers 1095 and 1096 (see Table 4 for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because rps4 and sufB share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of trnT , which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) ( trnR(ACG) *) and the 5' truncated rrl ( 'rrl ) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two trnT genes ( trnT -1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each trnT is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.
    Figure Legend Snippet: The rRNA/tRNA gene cluster of Plasmodium chabaudi chabaudi CB plastid DNA. (A) Part of the Pcc CB plastid DNA was amplified by PCR from total parasite DNA with the primers 1095 and 1096 (see Table 4 for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because rps4 and sufB share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of trnT , which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) ( trnR(ACG) *) and the 5' truncated rrl ( 'rrl ) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two trnT genes ( trnT -1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each trnT is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.

    Techniques Used: Periodic Counter-current Chromatography, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    Unique forms of the plastid DNA of Plasmodium chabaudi chabaudi CB and their proposed origin. Schematic diagram of the two forms of the plastid DNA of Pcc CB. Each gene is represented with a box color-coded in white (one specifying a protein), blue (rRNA gene), yellow (tRNA gene) or red (a pseudogene). The colour of the name of each gene represents the transcription direction (black, clockwise; red, counter-clockwise). The bulge connecting two parts of the trnL (UAA) coding sequence indicates the intron. The nucleotide sequences of the complete Pcc CB plastid DNA in forms A (A) and B (B) were deposited to the DDBJ/EMBL/GenBank databases with the accession numbers HF563595 and HF563596, respectively.
    Figure Legend Snippet: Unique forms of the plastid DNA of Plasmodium chabaudi chabaudi CB and their proposed origin. Schematic diagram of the two forms of the plastid DNA of Pcc CB. Each gene is represented with a box color-coded in white (one specifying a protein), blue (rRNA gene), yellow (tRNA gene) or red (a pseudogene). The colour of the name of each gene represents the transcription direction (black, clockwise; red, counter-clockwise). The bulge connecting two parts of the trnL (UAA) coding sequence indicates the intron. The nucleotide sequences of the complete Pcc CB plastid DNA in forms A (A) and B (B) were deposited to the DDBJ/EMBL/GenBank databases with the accession numbers HF563595 and HF563596, respectively.

    Techniques Used: Periodic Counter-current Chromatography, Sequencing

    Proposed origin of the unique Pcc CB plastid DNA. An intramolecular recombination event probably between trnI and trnV caused partial deletion of one of the two IR units in the ancestral DNA molecule (thick black arrow). The chimeric trnI / trnV in the intermediate molecule (1) was lost probably because it was a pseudogene. Subsequent truncation of the 5' region of rrl in the affected IR unit (thick blue arrow) and degradation of the trnR(ACG) in the other IR unit (thick red arrow) results in the Pcc plastid DNA. The order of these two events as well as which intermediate molecule (2) was generated in this process, are unknown. Finally, differentiation between trnM-1 and -2 as well as between trnT-1 and -2 occurred and these resulted in the Pcc CB plastid DNA. Switching between forms A and B could have happened at any point of this process, and may happen frequently as suggested by the fact that the apparent molar ratio between the two forms is 1∶1. Genes are color-coded as in panels A and B, and those changing/changed are indicated with a green circle.
    Figure Legend Snippet: Proposed origin of the unique Pcc CB plastid DNA. An intramolecular recombination event probably between trnI and trnV caused partial deletion of one of the two IR units in the ancestral DNA molecule (thick black arrow). The chimeric trnI / trnV in the intermediate molecule (1) was lost probably because it was a pseudogene. Subsequent truncation of the 5' region of rrl in the affected IR unit (thick blue arrow) and degradation of the trnR(ACG) in the other IR unit (thick red arrow) results in the Pcc plastid DNA. The order of these two events as well as which intermediate molecule (2) was generated in this process, are unknown. Finally, differentiation between trnM-1 and -2 as well as between trnT-1 and -2 occurred and these resulted in the Pcc CB plastid DNA. Switching between forms A and B could have happened at any point of this process, and may happen frequently as suggested by the fact that the apparent molar ratio between the two forms is 1∶1. Genes are color-coded as in panels A and B, and those changing/changed are indicated with a green circle.

    Techniques Used: Periodic Counter-current Chromatography, Generated

    3) Product Images from "The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi"

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061778

    The rRNA/tRNA gene cluster of Plasmodium chabaudi chabaudi CB plastid DNA. (A) Part of the Pcc CB plastid DNA was amplified by PCR from total parasite DNA with the primers 1095 and 1096 (see Table 4 for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because rps4 and sufB share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of trnT , which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) ( trnR(ACG) *) and the 5' truncated rrl ( 'rrl ) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two trnT genes ( trnT -1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each trnT is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.
    Figure Legend Snippet: The rRNA/tRNA gene cluster of Plasmodium chabaudi chabaudi CB plastid DNA. (A) Part of the Pcc CB plastid DNA was amplified by PCR from total parasite DNA with the primers 1095 and 1096 (see Table 4 for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because rps4 and sufB share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of trnT , which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) ( trnR(ACG) *) and the 5' truncated rrl ( 'rrl ) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two trnT genes ( trnT -1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each trnT is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.

    Techniques Used: Periodic Counter-current Chromatography, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    Unique forms of the plastid DNA of Plasmodium chabaudi chabaudi CB and their proposed origin. Schematic diagram of the two forms of the plastid DNA of Pcc CB. Each gene is represented with a box color-coded in white (one specifying a protein), blue (rRNA gene), yellow (tRNA gene) or red (a pseudogene). The colour of the name of each gene represents the transcription direction (black, clockwise; red, counter-clockwise). The bulge connecting two parts of the trnL (UAA) coding sequence indicates the intron. The nucleotide sequences of the complete Pcc CB plastid DNA in forms A (A) and B (B) were deposited to the DDBJ/EMBL/GenBank databases with the accession numbers HF563595 and HF563596, respectively.
    Figure Legend Snippet: Unique forms of the plastid DNA of Plasmodium chabaudi chabaudi CB and their proposed origin. Schematic diagram of the two forms of the plastid DNA of Pcc CB. Each gene is represented with a box color-coded in white (one specifying a protein), blue (rRNA gene), yellow (tRNA gene) or red (a pseudogene). The colour of the name of each gene represents the transcription direction (black, clockwise; red, counter-clockwise). The bulge connecting two parts of the trnL (UAA) coding sequence indicates the intron. The nucleotide sequences of the complete Pcc CB plastid DNA in forms A (A) and B (B) were deposited to the DDBJ/EMBL/GenBank databases with the accession numbers HF563595 and HF563596, respectively.

    Techniques Used: Periodic Counter-current Chromatography, Sequencing

    4) Product Images from "High Resolution Detection and Analysis of CpG Dinucleotides Methylation Using MBD-Seq Technology"

    Article Title: High Resolution Detection and Analysis of CpG Dinucleotides Methylation Using MBD-Seq Technology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022226

    Overview of experimental design and analysis. A. Major steps of MBD-seq experiment. Methylated DNA was enriched by recombinant MBD2 protein. Different fractions of enriched DNA fragments were eluted under three different salt concentrations (500 mM, 1000 mM, 2000 mM). Together with unenriched fraction, four DNA fractions were then used to construct four standard fragment libraries. Libraries were sequenced using SOLiD™ 3 Analyzer and the resulting tags were aligned to the human genome using the Bowtie mapping software. B. BALM algorithm includes a step of initial scanning target sites using a tag shifting method; followed by modeling tag distribution around target sites as a BALM. Then scan the genome for enriched regions and predict target sites by maximizing the likelihood of the given tags within the enriched regions. BIC is used to determine the number of target sites.
    Figure Legend Snippet: Overview of experimental design and analysis. A. Major steps of MBD-seq experiment. Methylated DNA was enriched by recombinant MBD2 protein. Different fractions of enriched DNA fragments were eluted under three different salt concentrations (500 mM, 1000 mM, 2000 mM). Together with unenriched fraction, four DNA fractions were then used to construct four standard fragment libraries. Libraries were sequenced using SOLiD™ 3 Analyzer and the resulting tags were aligned to the human genome using the Bowtie mapping software. B. BALM algorithm includes a step of initial scanning target sites using a tag shifting method; followed by modeling tag distribution around target sites as a BALM. Then scan the genome for enriched regions and predict target sites by maximizing the likelihood of the given tags within the enriched regions. BIC is used to determine the number of target sites.

    Techniques Used: Methylation, Recombinant, Construct, Software

    5) Product Images from "STR profiling and Copy Number Variation analysis on single, preserved cells using current Whole Genome Amplification methods"

    Article Title: STR profiling and Copy Number Variation analysis on single, preserved cells using current Whole Genome Amplification methods

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17525-5

    Experimental design. Cells from the Loucy cell line were preserved for 24 hours in Cell-Free DNA BCT reagent. Samples consisting of 1- or 3-cells were isolated from this fixed cell suspension for each WGA method. Ampli1, DOPlify, PicoPLEX and REPLI-g were used for amplification, followed by Illumina PCR-Free library preparation and next generation sequencing. In parallel, STR-PCR and capillary electrophoresis was performed on all samples, including a bulk sample from the cell line.
    Figure Legend Snippet: Experimental design. Cells from the Loucy cell line were preserved for 24 hours in Cell-Free DNA BCT reagent. Samples consisting of 1- or 3-cells were isolated from this fixed cell suspension for each WGA method. Ampli1, DOPlify, PicoPLEX and REPLI-g were used for amplification, followed by Illumina PCR-Free library preparation and next generation sequencing. In parallel, STR-PCR and capillary electrophoresis was performed on all samples, including a bulk sample from the cell line.

    Techniques Used: Isolation, Whole Genome Amplification, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Electrophoresis

    6) Product Images from "Prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling lens fiber differentiation and gene expression"

    Article Title: Prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling lens fiber differentiation and gene expression

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.127860

    Prox1 cKO lenses exhibit a decrease in the expression of three FGFRs and their downstream signaling. (A) qRT-PCR revealed significant decreases in the expression of Fgfr3 ( P =0.01), the atypical receptor Fgfrl1 ( P =0.04), and the FGF co-receptor Lctl ( P =0.03) in E14.5 Prox1 cKO lenses; * P
    Figure Legend Snippet: Prox1 cKO lenses exhibit a decrease in the expression of three FGFRs and their downstream signaling. (A) qRT-PCR revealed significant decreases in the expression of Fgfr3 ( P =0.01), the atypical receptor Fgfrl1 ( P =0.04), and the FGF co-receptor Lctl ( P =0.03) in E14.5 Prox1 cKO lenses; * P

    Techniques Used: Expressing, Quantitative RT-PCR

    LF marker expression decreases in Prox1 cKO. (A) qRT-PCR of E14.5 Prox1 cKO lenses compared with WT. * P ≤0.01 by nested ANOVA. αA-crystallin ( Cryaa ; P =0.007), βA1-crystallin ( Cryba1 ; P =0.001), βB1-crystallin ( Crybb1 ; P =0.002), γC-crystallin ( Crygc ; P =0.0001) and aquaporin 0 ( P =0.004) mRNA levels are reduced in Prox1 cKO lenses. Error bars indicate s.d.; n =3. (B,E) β-crystallin protein (brown) localizes to WT LFs at E13.5 (B), but this is reduced in Prox1 cKO lenses (E). (C,F) γ-Crystallin protein (green) is found in elongated LFs in the E13.5 WT lens (C), but its levels are reduced in the Prox1 cKO lens (F). (D,G) Aquaporin 0 protein (red) is found in E13.5 WT LFs (D), whereas little is detected in Prox1 cKO lenses (G). (B,E) Blue, Hematoxylin. (C,D,F,G) Blue, Draq5 (DNA). a, anterior eye; p, posterior eye; r, retina; e, lens epithelium; f, LFs. Scale bars: 200 µm in B,E; 100 µm in C,D,F,G.
    Figure Legend Snippet: LF marker expression decreases in Prox1 cKO. (A) qRT-PCR of E14.5 Prox1 cKO lenses compared with WT. * P ≤0.01 by nested ANOVA. αA-crystallin ( Cryaa ; P =0.007), βA1-crystallin ( Cryba1 ; P =0.001), βB1-crystallin ( Crybb1 ; P =0.002), γC-crystallin ( Crygc ; P =0.0001) and aquaporin 0 ( P =0.004) mRNA levels are reduced in Prox1 cKO lenses. Error bars indicate s.d.; n =3. (B,E) β-crystallin protein (brown) localizes to WT LFs at E13.5 (B), but this is reduced in Prox1 cKO lenses (E). (C,F) γ-Crystallin protein (green) is found in elongated LFs in the E13.5 WT lens (C), but its levels are reduced in the Prox1 cKO lens (F). (D,G) Aquaporin 0 protein (red) is found in E13.5 WT LFs (D), whereas little is detected in Prox1 cKO lenses (G). (B,E) Blue, Hematoxylin. (C,D,F,G) Blue, Draq5 (DNA). a, anterior eye; p, posterior eye; r, retina; e, lens epithelium; f, LFs. Scale bars: 200 µm in B,E; 100 µm in C,D,F,G.

    Techniques Used: Marker, Expressing, Quantitative RT-PCR

    Prox1 cKO lenses have a normal distribution of epithelial markers. (A,B) E-cadherin (red) is expressed in the epithelium and absent at the transition zone of both WT (A) and Prox1 cKO (B) E13.5 lenses. (C,D) The transcription factor Pax6 (red) is preferentially localized to the lens epithelium and reduced at the transition zones of both WT (C) and Prox1 cKO (D) lenses at E14.5. Blue, Draq5 (DNA). a, anterior eye; p, posterior eye; e, lens epithelium; f, LFs. Scale bar: 100 µm.
    Figure Legend Snippet: Prox1 cKO lenses have a normal distribution of epithelial markers. (A,B) E-cadherin (red) is expressed in the epithelium and absent at the transition zone of both WT (A) and Prox1 cKO (B) E13.5 lenses. (C,D) The transcription factor Pax6 (red) is preferentially localized to the lens epithelium and reduced at the transition zones of both WT (C) and Prox1 cKO (D) lenses at E14.5. Blue, Draq5 (DNA). a, anterior eye; p, posterior eye; e, lens epithelium; f, LFs. Scale bar: 100 µm.

    Techniques Used:

    7) Product Images from "High Resolution Detection and Analysis of CpG Dinucleotides Methylation Using MBD-Seq Technology"

    Article Title: High Resolution Detection and Analysis of CpG Dinucleotides Methylation Using MBD-Seq Technology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022226

    Overview of experimental design and analysis. A. Major steps of MBD-seq experiment. Methylated DNA was enriched by recombinant MBD2 protein. Different fractions of enriched DNA fragments were eluted under three different salt concentrations (500 mM, 1000 mM, 2000 mM). Together with unenriched fraction, four DNA fractions were then used to construct four standard fragment libraries. Libraries were sequenced using SOLiD™ 3 Analyzer and the resulting tags were aligned to the human genome using the Bowtie mapping software. B. BALM algorithm includes a step of initial scanning target sites using a tag shifting method; followed by modeling tag distribution around target sites as a BALM. Then scan the genome for enriched regions and predict target sites by maximizing the likelihood of the given tags within the enriched regions. BIC is used to determine the number of target sites.
    Figure Legend Snippet: Overview of experimental design and analysis. A. Major steps of MBD-seq experiment. Methylated DNA was enriched by recombinant MBD2 protein. Different fractions of enriched DNA fragments were eluted under three different salt concentrations (500 mM, 1000 mM, 2000 mM). Together with unenriched fraction, four DNA fractions were then used to construct four standard fragment libraries. Libraries were sequenced using SOLiD™ 3 Analyzer and the resulting tags were aligned to the human genome using the Bowtie mapping software. B. BALM algorithm includes a step of initial scanning target sites using a tag shifting method; followed by modeling tag distribution around target sites as a BALM. Then scan the genome for enriched regions and predict target sites by maximizing the likelihood of the given tags within the enriched regions. BIC is used to determine the number of target sites.

    Techniques Used: Methylation, Recombinant, Construct, Software

    Related Articles

    Transfection:

    Article Title: CRISPR-Cas3 induces broad and unidirectional genome editing in human cells
    Article Snippet: .. Deep sequencing for on- and off-target analysis For WGS, genomic DNA was extracted from transfected 293T cells and sheared using a Covaris ultrasonicator. .. After the preparation of a DNA library with the TruSeq DNA PCR-Free LT Library Prep Kit (Illumina), genomic sequence analysis was performed by HiSeq X (2 × 150 bp) according to the standard procedure at Takara Bio.

    Whole Genome Amplification:

    Article Title: Exploring DNA quality of single cells for genome analysis with simultaneous whole-genome amplification
    Article Snippet: .. Sequencing For DNA fragmentation, we used 2 μg genomic DNA or WGA DNA (without subsequent purification of WGA DNA) and the Covaris® S220 instrument. .. After fragmentation, DNA was purified using the MinElute® PCR Purification Kit (QIAGEN) according to the protocol in the kit handbook.

    Next-Generation Sequencing:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Infection:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Mouse Assay:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Sequencing:

    Article Title: Transcriptomics of diapause in an isogenic self-fertilizing vertebrate
    Article Snippet: .. Purified genomic DNA of Kmar FDS08 from our previous study was randomly selected for whole genome sequencing (WGS) (Additional file : Table S5) [ ] and sheared using Adaptive Focused Acoustics™ S2 (Covaris Inc., Woburn, MA). .. Libraries were constructed using NEXTflex™ DNA Sequencing Kit (Bioo Scientific Corp., Austin, TX).

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Article Title: Exploring DNA quality of single cells for genome analysis with simultaneous whole-genome amplification
    Article Snippet: .. Sequencing For DNA fragmentation, we used 2 μg genomic DNA or WGA DNA (without subsequent purification of WGA DNA) and the Covaris® S220 instrument. .. After fragmentation, DNA was purified using the MinElute® PCR Purification Kit (QIAGEN) according to the protocol in the kit handbook.

    Article Title: CRISPR-Cas3 induces broad and unidirectional genome editing in human cells
    Article Snippet: .. Deep sequencing for on- and off-target analysis For WGS, genomic DNA was extracted from transfected 293T cells and sheared using a Covaris ultrasonicator. .. After the preparation of a DNA library with the TruSeq DNA PCR-Free LT Library Prep Kit (Illumina), genomic sequence analysis was performed by HiSeq X (2 × 150 bp) according to the standard procedure at Takara Bio.

    DNA Sequencing:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Periodic Counter-current Chromatography:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Polymerase Chain Reaction:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Article Title: Advanced Whole Genome Sequencing Using a Complete PCR-free Massively Parallel Sequencing (MPS) Workflow
    Article Snippet: .. For acoustic PCR-free libraries, genomic DNA was fragmented to 100-600 bp with peak size at 350-400 bp using LE220 Ultrasonicator (Covaris, Woburn, MA, USA). .. For FS PCR-free libraries, genomic DNA was fragmented to 100-1000 bp with peak size at 350-475 bp using enzymatic shearing method.

    Purification:

    Article Title: Transcriptomics of diapause in an isogenic self-fertilizing vertebrate
    Article Snippet: .. Purified genomic DNA of Kmar FDS08 from our previous study was randomly selected for whole genome sequencing (WGS) (Additional file : Table S5) [ ] and sheared using Adaptive Focused Acoustics™ S2 (Covaris Inc., Woburn, MA). .. Libraries were constructed using NEXTflex™ DNA Sequencing Kit (Bioo Scientific Corp., Austin, TX).

    Article Title: Exploring DNA quality of single cells for genome analysis with simultaneous whole-genome amplification
    Article Snippet: .. Sequencing For DNA fragmentation, we used 2 μg genomic DNA or WGA DNA (without subsequent purification of WGA DNA) and the Covaris® S220 instrument. .. After fragmentation, DNA was purified using the MinElute® PCR Purification Kit (QIAGEN) according to the protocol in the kit handbook.

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