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    10X Genomics sequencing platforms
    A. L4 larvae (F1 generation) from a parental founder strain (P0) were individually picked onto NGM plates and allowed to self-fertilize prior to picking individual L4 larvae of the next generation (F2) from each F1 plate. This process was repeated until clonal lines reached generation F20 or F40. Clonal lines were then allowed to expand, harvested, and prepared for whole genome <t>sequencing</t> (Materials and Methods). B. Mutation types and their location on the 6 C . elegans chromosomes (I-V and X) across all wild-type samples and mutation classes. The height of the white bars corresponds to the length of the respective C . elegans chromosome. Single nucleotide variants are indicated by a dot, dinucleotide variants (DNVs) by a square, indels divided in deletions (D) and insertions (I) by a triangle, and structural variants (SVs) by a line. C. Average number of heterozygous mutations in the N2 wild-type genome per generation across all mutation classes and types. Single nucleotide variants are shown in the context of their 5’ and 3’ base. Grey bars denote 95% credible intervals for the number of mutations in each type. “Complex indels” class denotes deletions with insertions. Data for N2 was previously shown in ( Fig 1C) . Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).
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    1) Product Images from "Protection of the C . elegans germ cell genome depends on diverse DNA repair pathways during normal proliferation"

    Article Title: Protection of the C . elegans germ cell genome depends on diverse DNA repair pathways during normal proliferation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0250291

    A. L4 larvae (F1 generation) from a parental founder strain (P0) were individually picked onto NGM plates and allowed to self-fertilize prior to picking individual L4 larvae of the next generation (F2) from each F1 plate. This process was repeated until clonal lines reached generation F20 or F40. Clonal lines were then allowed to expand, harvested, and prepared for whole genome sequencing (Materials and Methods). B. Mutation types and their location on the 6 C . elegans chromosomes (I-V and X) across all wild-type samples and mutation classes. The height of the white bars corresponds to the length of the respective C . elegans chromosome. Single nucleotide variants are indicated by a dot, dinucleotide variants (DNVs) by a square, indels divided in deletions (D) and insertions (I) by a triangle, and structural variants (SVs) by a line. C. Average number of heterozygous mutations in the N2 wild-type genome per generation across all mutation classes and types. Single nucleotide variants are shown in the context of their 5’ and 3’ base. Grey bars denote 95% credible intervals for the number of mutations in each type. “Complex indels” class denotes deletions with insertions. Data for N2 was previously shown in ( Fig 1C) . Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).
    Figure Legend Snippet: A. L4 larvae (F1 generation) from a parental founder strain (P0) were individually picked onto NGM plates and allowed to self-fertilize prior to picking individual L4 larvae of the next generation (F2) from each F1 plate. This process was repeated until clonal lines reached generation F20 or F40. Clonal lines were then allowed to expand, harvested, and prepared for whole genome sequencing (Materials and Methods). B. Mutation types and their location on the 6 C . elegans chromosomes (I-V and X) across all wild-type samples and mutation classes. The height of the white bars corresponds to the length of the respective C . elegans chromosome. Single nucleotide variants are indicated by a dot, dinucleotide variants (DNVs) by a square, indels divided in deletions (D) and insertions (I) by a triangle, and structural variants (SVs) by a line. C. Average number of heterozygous mutations in the N2 wild-type genome per generation across all mutation classes and types. Single nucleotide variants are shown in the context of their 5’ and 3’ base. Grey bars denote 95% credible intervals for the number of mutations in each type. “Complex indels” class denotes deletions with insertions. Data for N2 was previously shown in ( Fig 1C) . Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).

    Techniques Used: Sequencing, Mutagenesis

    Mutation rates are shown as number of heterozygous mutations per generation for N2 wild-type (WT), and mutants used in this study grouped by the major DNA repair pathway they contribute to; direct damage reversal (DR), base excision repair (BER), nucleotide excision repair (NER), DNA double-strand break repair (DSBR), translesion synthesis (TLS), crosslink repair (ICLR), spindle assembly checkpoint (SAC), apoptosis, and mismatch repair (MMR). Base substitutions are shown in red (top), indels in green (center) and structural variants in blue (bottom). Dotted lines denote the mutation rates for wild-type. Error bars show the 95% confidence intervals; large dots represent variants with 2-fold increased or decreased mutation rates over N2 wild-type which are statistically significant with a false discovery rate (FDR) below 5%. All CIs which extend below the lower edge of the plot have zero as their lower border. Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).
    Figure Legend Snippet: Mutation rates are shown as number of heterozygous mutations per generation for N2 wild-type (WT), and mutants used in this study grouped by the major DNA repair pathway they contribute to; direct damage reversal (DR), base excision repair (BER), nucleotide excision repair (NER), DNA double-strand break repair (DSBR), translesion synthesis (TLS), crosslink repair (ICLR), spindle assembly checkpoint (SAC), apoptosis, and mismatch repair (MMR). Base substitutions are shown in red (top), indels in green (center) and structural variants in blue (bottom). Dotted lines denote the mutation rates for wild-type. Error bars show the 95% confidence intervals; large dots represent variants with 2-fold increased or decreased mutation rates over N2 wild-type which are statistically significant with a false discovery rate (FDR) below 5%. All CIs which extend below the lower edge of the plot have zero as their lower border. Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).

    Techniques Used: Mutagenesis, Translesion Synthesis, Sequencing

    A. Mutational signatures of BER, NER, and DR mutants that display statistically significantly different mutation spectra than wild-type shown as the number of mutations per generation across all mutation classes. Underscores (bold coloured bars) below each mutation profile indicate mutation types where the total mutation numbers are different from wild-type, three stars indicate genotypes with significantly different rates of substitutions, indels or SVs compared to those in wild-type (FDR < 5%). Single nucleotide variants are shown in the context of their 5’ and 3’ base context. B. Number of mutations of all classes shown for each individual sequenced line of the indicated genotype and generation. The four sequenced wild-type P0 lines reflect the variance present in initial generations. Mutations are shown cumulatively with mutations present in generation F20 included in F40. C. Mutation types of all classes and their location on the 6 C . elegans chromosomes (I-V and X) observed across agt-2 mutant lines. The height of the white bars corresponds to the length of each individual chromosome. Single nucleotide variants (SNVs) are indicated by a dot, dinucleotide variants (DNVs) by a square, indels divided in deletions (D), insertions (I), and deletions with insertions (DI) by a triangle, and structural variants (SVs) by a line. Clustered mutations that are present within a single agt-2 line are depicted by enlarged bold symbols. An analysis of brca-1 , him-6 and smc-6 swas previously shown in . Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).
    Figure Legend Snippet: A. Mutational signatures of BER, NER, and DR mutants that display statistically significantly different mutation spectra than wild-type shown as the number of mutations per generation across all mutation classes. Underscores (bold coloured bars) below each mutation profile indicate mutation types where the total mutation numbers are different from wild-type, three stars indicate genotypes with significantly different rates of substitutions, indels or SVs compared to those in wild-type (FDR < 5%). Single nucleotide variants are shown in the context of their 5’ and 3’ base context. B. Number of mutations of all classes shown for each individual sequenced line of the indicated genotype and generation. The four sequenced wild-type P0 lines reflect the variance present in initial generations. Mutations are shown cumulatively with mutations present in generation F20 included in F40. C. Mutation types of all classes and their location on the 6 C . elegans chromosomes (I-V and X) observed across agt-2 mutant lines. The height of the white bars corresponds to the length of each individual chromosome. Single nucleotide variants (SNVs) are indicated by a dot, dinucleotide variants (DNVs) by a square, indels divided in deletions (D), insertions (I), and deletions with insertions (DI) by a triangle, and structural variants (SVs) by a line. Clustered mutations that are present within a single agt-2 line are depicted by enlarged bold symbols. An analysis of brca-1 , him-6 and smc-6 swas previously shown in . Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).

    Techniques Used: Mutagenesis, Sequencing

    A. Mutational signatures of DSBR mutants that exhibited statistically significant different mutation rates to wild-type displayed in numbers of mutations per generation. Bold coloured bars denote individual mutation classes where the number of mutations is different from wild-type, an underscore below each mutation profile indicates mutation types with total mutation numbers different from wild-type, and three stars indicate genotypes which have rates of substitutions, indels or SVs significantly different compared to wild-type (FDR < 5%). B. Estimated composition of structural variants per generation as estimated for wild-type and DNA repair mutants with elevated SV rates. C. Size distributions of tandem duplications (top, pink) and deletions (bottom, green) across wild-type and mutants with elevated SV rates. D. Clustering of mutations in DNA repair deficient mutants. Grey dots reflect the average proportions of clustered mutations. Error bars denote 95% confidence intervals. Mutants with a significantly different propensity for mutation clustering from wild-type (dotted black line) are shown and highlighted in red. ‘Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).
    Figure Legend Snippet: A. Mutational signatures of DSBR mutants that exhibited statistically significant different mutation rates to wild-type displayed in numbers of mutations per generation. Bold coloured bars denote individual mutation classes where the number of mutations is different from wild-type, an underscore below each mutation profile indicates mutation types with total mutation numbers different from wild-type, and three stars indicate genotypes which have rates of substitutions, indels or SVs significantly different compared to wild-type (FDR < 5%). B. Estimated composition of structural variants per generation as estimated for wild-type and DNA repair mutants with elevated SV rates. C. Size distributions of tandem duplications (top, pink) and deletions (bottom, green) across wild-type and mutants with elevated SV rates. D. Clustering of mutations in DNA repair deficient mutants. Grey dots reflect the average proportions of clustered mutations. Error bars denote 95% confidence intervals. Mutants with a significantly different propensity for mutation clustering from wild-type (dotted black line) are shown and highlighted in red. ‘Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).

    Techniques Used: Mutagenesis, Sequencing

    A. Mutational signatures of TLS and ICLR mutants that exhibited statistically significant differences to wild-type mutation rates displayed in numbers of mutations per generation. Same layout as . B . Proportion of indels (brown) and SVs (black) in G-rich regions in wild-type and across genotypes with elevated rates of SVs. Dotted line represents the proportion of variants falling into these regions as expected by chance. C . Tandem duplications (TDs) in helq-1 mutants. An analysis of rev-3 was previously shown in . Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).
    Figure Legend Snippet: A. Mutational signatures of TLS and ICLR mutants that exhibited statistically significant differences to wild-type mutation rates displayed in numbers of mutations per generation. Same layout as . B . Proportion of indels (brown) and SVs (black) in G-rich regions in wild-type and across genotypes with elevated rates of SVs. Dotted line represents the proportion of variants falling into these regions as expected by chance. C . Tandem duplications (TDs) in helq-1 mutants. An analysis of rev-3 was previously shown in . Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).

    Techniques Used: Mutagenesis, Sequencing

    A. Mutational signatures of mutants that exhibited statistically significant differences to wild-type mutation rates. The chromosomes on which respective genes are located are indicated in superscript following each mutant name. Layout as . B. Proportion of SVs with breakpoints into subtelomeric regions across wild-type and mutants that exhibit elevated SV rates. Dotted lines represent the fraction of variants expected to occur in subtelomeric regions by chance. C . Examples of subtelomeric structural variants in atm-1 mutants. D. Quantification of mutation burden in indicated DNA repair mutants for initial generations and F20 and F40 generations as shown. Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).
    Figure Legend Snippet: A. Mutational signatures of mutants that exhibited statistically significant differences to wild-type mutation rates. The chromosomes on which respective genes are located are indicated in superscript following each mutant name. Layout as . B. Proportion of SVs with breakpoints into subtelomeric regions across wild-type and mutants that exhibit elevated SV rates. Dotted lines represent the fraction of variants expected to occur in subtelomeric regions by chance. C . Examples of subtelomeric structural variants in atm-1 mutants. D. Quantification of mutation burden in indicated DNA repair mutants for initial generations and F20 and F40 generations as shown. Information related to the 528 whole genome sequencing WGS primary-source datasets (56 deposited in this study, 472 deposited in (Suppl Data 1 and Supple Note 1 of can be found in ).

    Techniques Used: Mutagenesis, Sequencing

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    Single Cell:

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    Marker:

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    Expressing:

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    Plasmidsaurus nanopore sequencing platform
    Confirmation of the mutant E. chaffeensis by PCR and whole-genome <t>sequencing.</t> ( A ) Schematic view of the duplex RNA strand formation of asRNA with mRNA of ECH_1143 . The entire predicted asRNA and sense RNA forming hybrids are presented in the image. ( B ) PCR analysis confirming the mutation generation. Primers annealing upstream (P1) and downstream (P5) of the mutation insertion region (genomic coordinates identified) were used to amplify DNA segments by PCR I, which were then resolved on an agarose gel; the expected larger 4.77 kb product was evident from the mutant genomic DNA (M) compared to the smaller 2.07 kb from wild-type genomic DNA (W). (L, 1 kb plus DNA ladder). Similarly, PCR II using primers P1 and P2 targeted to the insertion region and genomic region upstream of the insertion region yielded the expected amplicon of 2.18 kb only from the mutant. ( C ) <t>Nanopore</t> whole-genome sequencing reads were used to assemble the genome of the antisense mutant. The insertion segment was identified at the anticipated region within the E. chaffeensis genome. ( D ) Transcriptional analysis confirming the ECH_1143 asRNA. The presence of asRNA was assessed by RT-PCR using RNA isolated from wild-type (W) and mutant (M) E. chaffeensis in the macrophage cell line and tick cell line. RNA samples were reverse transcribed to generate cDNA using primer P4, which was designed to anneal downstream of the asRNA sequence, which was expected to be present only in the asRNA mutant. The cDNA was used as the template for PCR amplification with primers P3 and P4 yielding the expected amplicon of 263 bp. Genomic DNA and RNA with no reverse transcription step from both wild-type and mutant E. chaffeensis were included as controls, along with a no-template reaction (−). DNA marker (1 kb Plus DNA ladder) (L) was used to identify specific size amplicon. The asRNA amplicon was detected in the cDNA from the mutant, but not in the cDNA from wild type. Mutant genomic DNA served as the positive control and similarly genomic DNA from wild-type E. chaffeensis served as a negative control along with a no-template negative control. RNA-only controls (with no reverse transcription steps) also yielded the anticipated negative results. ( E ) The impact of ECH_1143 antisense construct insertion on the RNA expression of upstream ( ECH_0230 ) and downstream ( ECH_0232 ) genes. Transcriptional analysis of RNA isolated from asRNA mutant (M) and wild-type (W) E. chaffeensis in macrophage cells was conducted by RT-PCR targeting ECH_0230 and ECH_0232 . RNA input was normalized based on 16S rRNA levels quantified by qRT-PCR. Transcript levels of the target genes were then assessed by semi-quantitative RT-PCR (30 cycles). Wild-type genomic DNA served as a positive control (+), and a no-template reaction was included as a negative control (−). DNA marker (1 kb Plus DNA ladder) (L) was used to verify the expected amplicon sizes. Expression of ECH_0230 and ECH_0232 was similar for wild-type and mutant E. chaffeensis .
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    sequencing platforms  (Pacific Biosciences)
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    Confirmation of the mutant E. chaffeensis by PCR and whole-genome <t>sequencing.</t> ( A ) Schematic view of the duplex RNA strand formation of asRNA with mRNA of ECH_1143 . The entire predicted asRNA and sense RNA forming hybrids are presented in the image. ( B ) PCR analysis confirming the mutation generation. Primers annealing upstream (P1) and downstream (P5) of the mutation insertion region (genomic coordinates identified) were used to amplify DNA segments by PCR I, which were then resolved on an agarose gel; the expected larger 4.77 kb product was evident from the mutant genomic DNA (M) compared to the smaller 2.07 kb from wild-type genomic DNA (W). (L, 1 kb plus DNA ladder). Similarly, PCR II using primers P1 and P2 targeted to the insertion region and genomic region upstream of the insertion region yielded the expected amplicon of 2.18 kb only from the mutant. ( C ) <t>Nanopore</t> whole-genome sequencing reads were used to assemble the genome of the antisense mutant. The insertion segment was identified at the anticipated region within the E. chaffeensis genome. ( D ) Transcriptional analysis confirming the ECH_1143 asRNA. The presence of asRNA was assessed by RT-PCR using RNA isolated from wild-type (W) and mutant (M) E. chaffeensis in the macrophage cell line and tick cell line. RNA samples were reverse transcribed to generate cDNA using primer P4, which was designed to anneal downstream of the asRNA sequence, which was expected to be present only in the asRNA mutant. The cDNA was used as the template for PCR amplification with primers P3 and P4 yielding the expected amplicon of 263 bp. Genomic DNA and RNA with no reverse transcription step from both wild-type and mutant E. chaffeensis were included as controls, along with a no-template reaction (−). DNA marker (1 kb Plus DNA ladder) (L) was used to identify specific size amplicon. The asRNA amplicon was detected in the cDNA from the mutant, but not in the cDNA from wild type. Mutant genomic DNA served as the positive control and similarly genomic DNA from wild-type E. chaffeensis served as a negative control along with a no-template negative control. RNA-only controls (with no reverse transcription steps) also yielded the anticipated negative results. ( E ) The impact of ECH_1143 antisense construct insertion on the RNA expression of upstream ( ECH_0230 ) and downstream ( ECH_0232 ) genes. Transcriptional analysis of RNA isolated from asRNA mutant (M) and wild-type (W) E. chaffeensis in macrophage cells was conducted by RT-PCR targeting ECH_0230 and ECH_0232 . RNA input was normalized based on 16S rRNA levels quantified by qRT-PCR. Transcript levels of the target genes were then assessed by semi-quantitative RT-PCR (30 cycles). Wild-type genomic DNA served as a positive control (+), and a no-template reaction was included as a negative control (−). DNA marker (1 kb Plus DNA ladder) (L) was used to verify the expected amplicon sizes. Expression of ECH_0230 and ECH_0232 was similar for wild-type and mutant E. chaffeensis .
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    aviti sequencing platform  (Element Biosciences Inc)
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    Element Biosciences Inc aviti sequencing platform
    Confirmation of the mutant E. chaffeensis by PCR and whole-genome <t>sequencing.</t> ( A ) Schematic view of the duplex RNA strand formation of asRNA with mRNA of ECH_1143 . The entire predicted asRNA and sense RNA forming hybrids are presented in the image. ( B ) PCR analysis confirming the mutation generation. Primers annealing upstream (P1) and downstream (P5) of the mutation insertion region (genomic coordinates identified) were used to amplify DNA segments by PCR I, which were then resolved on an agarose gel; the expected larger 4.77 kb product was evident from the mutant genomic DNA (M) compared to the smaller 2.07 kb from wild-type genomic DNA (W). (L, 1 kb plus DNA ladder). Similarly, PCR II using primers P1 and P2 targeted to the insertion region and genomic region upstream of the insertion region yielded the expected amplicon of 2.18 kb only from the mutant. ( C ) <t>Nanopore</t> whole-genome sequencing reads were used to assemble the genome of the antisense mutant. The insertion segment was identified at the anticipated region within the E. chaffeensis genome. ( D ) Transcriptional analysis confirming the ECH_1143 asRNA. The presence of asRNA was assessed by RT-PCR using RNA isolated from wild-type (W) and mutant (M) E. chaffeensis in the macrophage cell line and tick cell line. RNA samples were reverse transcribed to generate cDNA using primer P4, which was designed to anneal downstream of the asRNA sequence, which was expected to be present only in the asRNA mutant. The cDNA was used as the template for PCR amplification with primers P3 and P4 yielding the expected amplicon of 263 bp. Genomic DNA and RNA with no reverse transcription step from both wild-type and mutant E. chaffeensis were included as controls, along with a no-template reaction (−). DNA marker (1 kb Plus DNA ladder) (L) was used to identify specific size amplicon. The asRNA amplicon was detected in the cDNA from the mutant, but not in the cDNA from wild type. Mutant genomic DNA served as the positive control and similarly genomic DNA from wild-type E. chaffeensis served as a negative control along with a no-template negative control. RNA-only controls (with no reverse transcription steps) also yielded the anticipated negative results. ( E ) The impact of ECH_1143 antisense construct insertion on the RNA expression of upstream ( ECH_0230 ) and downstream ( ECH_0232 ) genes. Transcriptional analysis of RNA isolated from asRNA mutant (M) and wild-type (W) E. chaffeensis in macrophage cells was conducted by RT-PCR targeting ECH_0230 and ECH_0232 . RNA input was normalized based on 16S rRNA levels quantified by qRT-PCR. Transcript levels of the target genes were then assessed by semi-quantitative RT-PCR (30 cycles). Wild-type genomic DNA served as a positive control (+), and a no-template reaction was included as a negative control (−). DNA marker (1 kb Plus DNA ladder) (L) was used to verify the expected amplicon sizes. Expression of ECH_0230 and ECH_0232 was similar for wild-type and mutant E. chaffeensis .
    Aviti Sequencing Platform, supplied by Element Biosciences Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    miseq sequencing platform  (Illumina Inc)
    99
    Illumina Inc miseq sequencing platform
    Confirmation of the mutant E. chaffeensis by PCR and whole-genome <t>sequencing.</t> ( A ) Schematic view of the duplex RNA strand formation of asRNA with mRNA of ECH_1143 . The entire predicted asRNA and sense RNA forming hybrids are presented in the image. ( B ) PCR analysis confirming the mutation generation. Primers annealing upstream (P1) and downstream (P5) of the mutation insertion region (genomic coordinates identified) were used to amplify DNA segments by PCR I, which were then resolved on an agarose gel; the expected larger 4.77 kb product was evident from the mutant genomic DNA (M) compared to the smaller 2.07 kb from wild-type genomic DNA (W). (L, 1 kb plus DNA ladder). Similarly, PCR II using primers P1 and P2 targeted to the insertion region and genomic region upstream of the insertion region yielded the expected amplicon of 2.18 kb only from the mutant. ( C ) <t>Nanopore</t> whole-genome sequencing reads were used to assemble the genome of the antisense mutant. The insertion segment was identified at the anticipated region within the E. chaffeensis genome. ( D ) Transcriptional analysis confirming the ECH_1143 asRNA. The presence of asRNA was assessed by RT-PCR using RNA isolated from wild-type (W) and mutant (M) E. chaffeensis in the macrophage cell line and tick cell line. RNA samples were reverse transcribed to generate cDNA using primer P4, which was designed to anneal downstream of the asRNA sequence, which was expected to be present only in the asRNA mutant. The cDNA was used as the template for PCR amplification with primers P3 and P4 yielding the expected amplicon of 263 bp. Genomic DNA and RNA with no reverse transcription step from both wild-type and mutant E. chaffeensis were included as controls, along with a no-template reaction (−). DNA marker (1 kb Plus DNA ladder) (L) was used to identify specific size amplicon. The asRNA amplicon was detected in the cDNA from the mutant, but not in the cDNA from wild type. Mutant genomic DNA served as the positive control and similarly genomic DNA from wild-type E. chaffeensis served as a negative control along with a no-template negative control. RNA-only controls (with no reverse transcription steps) also yielded the anticipated negative results. ( E ) The impact of ECH_1143 antisense construct insertion on the RNA expression of upstream ( ECH_0230 ) and downstream ( ECH_0232 ) genes. Transcriptional analysis of RNA isolated from asRNA mutant (M) and wild-type (W) E. chaffeensis in macrophage cells was conducted by RT-PCR targeting ECH_0230 and ECH_0232 . RNA input was normalized based on 16S rRNA levels quantified by qRT-PCR. Transcript levels of the target genes were then assessed by semi-quantitative RT-PCR (30 cycles). Wild-type genomic DNA served as a positive control (+), and a no-template reaction was included as a negative control (−). DNA marker (1 kb Plus DNA ladder) (L) was used to verify the expected amplicon sizes. Expression of ECH_0230 and ECH_0232 was similar for wild-type and mutant E. chaffeensis .
    Miseq Sequencing Platform, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    plasmidsaurus sequencing platform  (Plasmidsaurus)
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    Plasmidsaurus plasmidsaurus sequencing platform
    (A) pFRIT4.0-DgRNA plasmid map. The two sgRNA cassettes are indicated (B) Genome positions of MBOVPG45_0215 ( mnuA ) and MBOVPG45_0690 (5’- nt ) targeted genes and respective location of the generated stop codons within genes. (C) Sanger sequencings of the targeted sites within MBOVPG45_0215 (left) and MBOVPG45_0690 (right) of an isolated mutant. The percentage of bases found in the isolated clones of transformants were determined from Sanger <t>sequencing</t> chromatograms using the Base editor processing tool from Han lab .
    Plasmidsaurus Sequencing Platform, supplied by Plasmidsaurus, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    generation long read sequencing platforms  (Pacific Biosciences)
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    <t>Single-cell</t> <t>long-read</t> <t>sequencing</t> (scLRS) enhances tumor cell resolution by integrating methods for detecting splicing alterations and genetic variants. Schematic representation of how scLRS can visualize and combine multiple information types within the same cells to improve tumor cell resolution. Created in BioRender. Byrne, A. (2026) https://BioRender.com/mrmobm6 .
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    <t>Single-cell</t> <t>long-read</t> <t>sequencing</t> (scLRS) enhances tumor cell resolution by integrating methods for detecting splicing alterations and genetic variants. Schematic representation of how scLRS can visualize and combine multiple information types within the same cells to improve tumor cell resolution. Created in BioRender. Byrne, A. (2026) https://BioRender.com/mrmobm6 .
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    <t>Single-cell</t> <t>long-read</t> <t>sequencing</t> (scLRS) enhances tumor cell resolution by integrating methods for detecting splicing alterations and genetic variants. Schematic representation of how scLRS can visualize and combine multiple information types within the same cells to improve tumor cell resolution. Created in BioRender. Byrne, A. (2026) https://BioRender.com/mrmobm6 .
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    Image Search Results


    Confirmation of the mutant E. chaffeensis by PCR and whole-genome sequencing. ( A ) Schematic view of the duplex RNA strand formation of asRNA with mRNA of ECH_1143 . The entire predicted asRNA and sense RNA forming hybrids are presented in the image. ( B ) PCR analysis confirming the mutation generation. Primers annealing upstream (P1) and downstream (P5) of the mutation insertion region (genomic coordinates identified) were used to amplify DNA segments by PCR I, which were then resolved on an agarose gel; the expected larger 4.77 kb product was evident from the mutant genomic DNA (M) compared to the smaller 2.07 kb from wild-type genomic DNA (W). (L, 1 kb plus DNA ladder). Similarly, PCR II using primers P1 and P2 targeted to the insertion region and genomic region upstream of the insertion region yielded the expected amplicon of 2.18 kb only from the mutant. ( C ) Nanopore whole-genome sequencing reads were used to assemble the genome of the antisense mutant. The insertion segment was identified at the anticipated region within the E. chaffeensis genome. ( D ) Transcriptional analysis confirming the ECH_1143 asRNA. The presence of asRNA was assessed by RT-PCR using RNA isolated from wild-type (W) and mutant (M) E. chaffeensis in the macrophage cell line and tick cell line. RNA samples were reverse transcribed to generate cDNA using primer P4, which was designed to anneal downstream of the asRNA sequence, which was expected to be present only in the asRNA mutant. The cDNA was used as the template for PCR amplification with primers P3 and P4 yielding the expected amplicon of 263 bp. Genomic DNA and RNA with no reverse transcription step from both wild-type and mutant E. chaffeensis were included as controls, along with a no-template reaction (−). DNA marker (1 kb Plus DNA ladder) (L) was used to identify specific size amplicon. The asRNA amplicon was detected in the cDNA from the mutant, but not in the cDNA from wild type. Mutant genomic DNA served as the positive control and similarly genomic DNA from wild-type E. chaffeensis served as a negative control along with a no-template negative control. RNA-only controls (with no reverse transcription steps) also yielded the anticipated negative results. ( E ) The impact of ECH_1143 antisense construct insertion on the RNA expression of upstream ( ECH_0230 ) and downstream ( ECH_0232 ) genes. Transcriptional analysis of RNA isolated from asRNA mutant (M) and wild-type (W) E. chaffeensis in macrophage cells was conducted by RT-PCR targeting ECH_0230 and ECH_0232 . RNA input was normalized based on 16S rRNA levels quantified by qRT-PCR. Transcript levels of the target genes were then assessed by semi-quantitative RT-PCR (30 cycles). Wild-type genomic DNA served as a positive control (+), and a no-template reaction was included as a negative control (−). DNA marker (1 kb Plus DNA ladder) (L) was used to verify the expected amplicon sizes. Expression of ECH_0230 and ECH_0232 was similar for wild-type and mutant E. chaffeensis .

    Journal: Journal of Bacteriology

    Article Title: A targeted mutational strategy aiding in generating antisense RNA to knockdown the Ehrlichia chaffeensis P28-outer membrane protein 19 expression

    doi: 10.1128/jb.00334-26

    Figure Lengend Snippet: Confirmation of the mutant E. chaffeensis by PCR and whole-genome sequencing. ( A ) Schematic view of the duplex RNA strand formation of asRNA with mRNA of ECH_1143 . The entire predicted asRNA and sense RNA forming hybrids are presented in the image. ( B ) PCR analysis confirming the mutation generation. Primers annealing upstream (P1) and downstream (P5) of the mutation insertion region (genomic coordinates identified) were used to amplify DNA segments by PCR I, which were then resolved on an agarose gel; the expected larger 4.77 kb product was evident from the mutant genomic DNA (M) compared to the smaller 2.07 kb from wild-type genomic DNA (W). (L, 1 kb plus DNA ladder). Similarly, PCR II using primers P1 and P2 targeted to the insertion region and genomic region upstream of the insertion region yielded the expected amplicon of 2.18 kb only from the mutant. ( C ) Nanopore whole-genome sequencing reads were used to assemble the genome of the antisense mutant. The insertion segment was identified at the anticipated region within the E. chaffeensis genome. ( D ) Transcriptional analysis confirming the ECH_1143 asRNA. The presence of asRNA was assessed by RT-PCR using RNA isolated from wild-type (W) and mutant (M) E. chaffeensis in the macrophage cell line and tick cell line. RNA samples were reverse transcribed to generate cDNA using primer P4, which was designed to anneal downstream of the asRNA sequence, which was expected to be present only in the asRNA mutant. The cDNA was used as the template for PCR amplification with primers P3 and P4 yielding the expected amplicon of 263 bp. Genomic DNA and RNA with no reverse transcription step from both wild-type and mutant E. chaffeensis were included as controls, along with a no-template reaction (−). DNA marker (1 kb Plus DNA ladder) (L) was used to identify specific size amplicon. The asRNA amplicon was detected in the cDNA from the mutant, but not in the cDNA from wild type. Mutant genomic DNA served as the positive control and similarly genomic DNA from wild-type E. chaffeensis served as a negative control along with a no-template negative control. RNA-only controls (with no reverse transcription steps) also yielded the anticipated negative results. ( E ) The impact of ECH_1143 antisense construct insertion on the RNA expression of upstream ( ECH_0230 ) and downstream ( ECH_0232 ) genes. Transcriptional analysis of RNA isolated from asRNA mutant (M) and wild-type (W) E. chaffeensis in macrophage cells was conducted by RT-PCR targeting ECH_0230 and ECH_0232 . RNA input was normalized based on 16S rRNA levels quantified by qRT-PCR. Transcript levels of the target genes were then assessed by semi-quantitative RT-PCR (30 cycles). Wild-type genomic DNA served as a positive control (+), and a no-template reaction was included as a negative control (−). DNA marker (1 kb Plus DNA ladder) (L) was used to verify the expected amplicon sizes. Expression of ECH_0230 and ECH_0232 was similar for wild-type and mutant E. chaffeensis .

    Article Snippet: Additionally, whole-genome sequencing was performed using mutant genomic DNA on Nanopore sequencing platform (Plasmidsaurus Inc., KY, USA) to independently confirm the mutation and to rule out any off-target insertions.

    Techniques: Mutagenesis, Sequencing, Agarose Gel Electrophoresis, Amplification, Reverse Transcription Polymerase Chain Reaction, Isolation, Reverse Transcription, Marker, Positive Control, Negative Control, Construct, RNA Expression, Quantitative RT-PCR, Expressing

    (A) pFRIT4.0-DgRNA plasmid map. The two sgRNA cassettes are indicated (B) Genome positions of MBOVPG45_0215 ( mnuA ) and MBOVPG45_0690 (5’- nt ) targeted genes and respective location of the generated stop codons within genes. (C) Sanger sequencings of the targeted sites within MBOVPG45_0215 (left) and MBOVPG45_0690 (right) of an isolated mutant. The percentage of bases found in the isolated clones of transformants were determined from Sanger sequencing chromatograms using the Base editor processing tool from Han lab .

    Journal: bioRxiv

    Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

    doi: 10.64898/2026.05.29.712936

    Figure Lengend Snippet: (A) pFRIT4.0-DgRNA plasmid map. The two sgRNA cassettes are indicated (B) Genome positions of MBOVPG45_0215 ( mnuA ) and MBOVPG45_0690 (5’- nt ) targeted genes and respective location of the generated stop codons within genes. (C) Sanger sequencings of the targeted sites within MBOVPG45_0215 (left) and MBOVPG45_0690 (right) of an isolated mutant. The percentage of bases found in the isolated clones of transformants were determined from Sanger sequencing chromatograms using the Base editor processing tool from Han lab .

    Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

    Techniques: Plasmid Preparation, Generated, Isolation, Mutagenesis, Clone Assay, Sequencing

    (A) Localisation of the 11 copies of IS Mbov1 within the genome of M. bovis PG45 strain. (B) Targeted regions of IS Mbov1 copies through double gRNA expression and the respective location of the generated stop codons (C) IS Mbov1 isoform sequence conservation of targeted regions (representation through muscle alignment). (D) gRNAs target sequences conservation within IS Mbov1 done through Web-logo . (E) Sanger sequencing of the targeted sites, Target 1 (left) and Target 2 (right) within an isolated mutant after three rounds of induction. Chromatograms were analysed using the Base editor processing tool from Han lab .

    Journal: bioRxiv

    Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

    doi: 10.64898/2026.05.29.712936

    Figure Lengend Snippet: (A) Localisation of the 11 copies of IS Mbov1 within the genome of M. bovis PG45 strain. (B) Targeted regions of IS Mbov1 copies through double gRNA expression and the respective location of the generated stop codons (C) IS Mbov1 isoform sequence conservation of targeted regions (representation through muscle alignment). (D) gRNAs target sequences conservation within IS Mbov1 done through Web-logo . (E) Sanger sequencing of the targeted sites, Target 1 (left) and Target 2 (right) within an isolated mutant after three rounds of induction. Chromatograms were analysed using the Base editor processing tool from Han lab .

    Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

    Techniques: Expressing, Generated, Sequencing, Isolation, Mutagenesis

    To obtain full deamination on all ISMbov1 coding sequences, three rounds of induction were necessary. An example of the profiles obtained after one, two or three rounds of deamination for one of the three clones is presented here. The first round was conducted as previous cultures were, with overnight induction, followed by gDNA extraction and Sanger screening. After primary analysis, which revealed that full deamination had not occurred, two supplementary passages under inducting conditions were performed and gDNA was extracted at each of them for PCR amplification and Sanger sequencing.

    Journal: bioRxiv

    Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

    doi: 10.64898/2026.05.29.712936

    Figure Lengend Snippet: To obtain full deamination on all ISMbov1 coding sequences, three rounds of induction were necessary. An example of the profiles obtained after one, two or three rounds of deamination for one of the three clones is presented here. The first round was conducted as previous cultures were, with overnight induction, followed by gDNA extraction and Sanger screening. After primary analysis, which revealed that full deamination had not occurred, two supplementary passages under inducting conditions were performed and gDNA was extracted at each of them for PCR amplification and Sanger sequencing.

    Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

    Techniques: Clone Assay, Extraction, Amplification, Sequencing

    (A) Targeted regions of MIP genes conducted with CRISPR-VQR variants. (B) Representation of Sanger sequencing of the targeted sites within MBOVPG45_0373 (left) or MBOVPG45_0376 (Right) within two isolated mutants. The black square indicates the used PAM sequence. Sanger sequencing chromatogram analysis was done using Base editor processing tool from Han lab .

    Journal: bioRxiv

    Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

    doi: 10.64898/2026.05.29.712936

    Figure Lengend Snippet: (A) Targeted regions of MIP genes conducted with CRISPR-VQR variants. (B) Representation of Sanger sequencing of the targeted sites within MBOVPG45_0373 (left) or MBOVPG45_0376 (Right) within two isolated mutants. The black square indicates the used PAM sequence. Sanger sequencing chromatogram analysis was done using Base editor processing tool from Han lab .

    Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

    Techniques: CRISPR, Sequencing, Isolation

    After recombination between the res sequences induced by the γδ resolvase, a 218 bp scar remains at the site of integration of the transposon. It includes the remaining res sequence (coloured in red) and inverted repeats (IR) at the extremities of the transposon (coloured in green).

    Journal: bioRxiv

    Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

    doi: 10.64898/2026.05.29.712936

    Figure Lengend Snippet: After recombination between the res sequences induced by the γδ resolvase, a 218 bp scar remains at the site of integration of the transposon. It includes the remaining res sequence (coloured in red) and inverted repeats (IR) at the extremities of the transposon (coloured in green).

    Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

    Techniques: Sequencing

    The multi-step process for the generation of deaminated-resolved-cured (DRC) mutants involves first the transformation of the mycoplasma, the integration of the transposon harbouring the CRISPR-BE and induction of its expression to obtain a deaminated mutant. PCR and Sanger sequencing screening is then used to select a deaminated mutant. Resolution of the transposon is then achieved by transformation with the pRES plasmid. Continued passages of isolated mutants under selection with tetracycline 5 µg.µL - are combined with DNA extraction and PCR amplification at each passage to verify the excision of the transposon cassette. Once excision is validated by PCR, gentamicin susceptibility is confirmed by culture in gentamicin selective media at 100 µg.mL - . Following confirmation, the isolated mutants are cultivated in non-selective media and subcloned until negative PCR amplification of a pRES region and inability to grow in selective tetracycline medium are obtained. Final verifications were done through Sanger sequencing of the deamination site and WGS to verify genomic scar presence.

    Journal: bioRxiv

    Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

    doi: 10.64898/2026.05.29.712936

    Figure Lengend Snippet: The multi-step process for the generation of deaminated-resolved-cured (DRC) mutants involves first the transformation of the mycoplasma, the integration of the transposon harbouring the CRISPR-BE and induction of its expression to obtain a deaminated mutant. PCR and Sanger sequencing screening is then used to select a deaminated mutant. Resolution of the transposon is then achieved by transformation with the pRES plasmid. Continued passages of isolated mutants under selection with tetracycline 5 µg.µL - are combined with DNA extraction and PCR amplification at each passage to verify the excision of the transposon cassette. Once excision is validated by PCR, gentamicin susceptibility is confirmed by culture in gentamicin selective media at 100 µg.mL - . Following confirmation, the isolated mutants are cultivated in non-selective media and subcloned until negative PCR amplification of a pRES region and inability to grow in selective tetracycline medium are obtained. Final verifications were done through Sanger sequencing of the deamination site and WGS to verify genomic scar presence.

    Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

    Techniques: Transformation Assay, CRISPR, Expressing, Mutagenesis, Sequencing, Plasmid Preparation, Isolation, Selection, DNA Extraction, Amplification

    WGS sequencing revealed the successful resolution of the transposon and indicated insertion locus of the employed CRISPR-BE was intragenic to the MBOVPG45_756 coding sequence. (A) Genome structure in the WT and in the resolved mutant. (B) Localisation of the transposon insertion site and targeted sites in the genome of M. bovis PG45.

    Journal: bioRxiv

    Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

    doi: 10.64898/2026.05.29.712936

    Figure Lengend Snippet: WGS sequencing revealed the successful resolution of the transposon and indicated insertion locus of the employed CRISPR-BE was intragenic to the MBOVPG45_756 coding sequence. (A) Genome structure in the WT and in the resolved mutant. (B) Localisation of the transposon insertion site and targeted sites in the genome of M. bovis PG45.

    Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

    Techniques: Sequencing, CRISPR, Mutagenesis

    In the case of the obtained MBOVPG45_0215 mutant with the pFrHog plasmid, the process to obtain a cured mutant is a condensed version of the previously described DRC pipeline. After transformation, a single round of induction is necessary to obtain the desired base-editing. PCR verifications for the presence of mutation and cultures in selective medium are used to follow the process. Curing of the plasmid is then obtained by passages in non-selective medium. Once both validation steps are validated, the isolated mutant is considered to be cured and can be reverified at the deamination site through PCR and Sanger sequencing.

    Journal: bioRxiv

    Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

    doi: 10.64898/2026.05.29.712936

    Figure Lengend Snippet: In the case of the obtained MBOVPG45_0215 mutant with the pFrHog plasmid, the process to obtain a cured mutant is a condensed version of the previously described DRC pipeline. After transformation, a single round of induction is necessary to obtain the desired base-editing. PCR verifications for the presence of mutation and cultures in selective medium are used to follow the process. Curing of the plasmid is then obtained by passages in non-selective medium. Once both validation steps are validated, the isolated mutant is considered to be cured and can be reverified at the deamination site through PCR and Sanger sequencing.

    Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

    Techniques: Mutagenesis, Plasmid Preparation, Transformation Assay, Biomarker Discovery, Isolation, Sequencing

    Representation of the percentage of targetable CDS by the CRISPR-BE or CRISPR-BE VQR tools compared to the total number of CDS in multiple mycoplasma species. Representation of the percentage of genes targetable within the first 10 th , 30 th , 50 th and whole coding sequence.

    Journal: bioRxiv

    Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

    doi: 10.64898/2026.05.29.712936

    Figure Lengend Snippet: Representation of the percentage of targetable CDS by the CRISPR-BE or CRISPR-BE VQR tools compared to the total number of CDS in multiple mycoplasma species. Representation of the percentage of genes targetable within the first 10 th , 30 th , 50 th and whole coding sequence.

    Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

    Techniques: CRISPR, Sequencing

    Single-cell long-read sequencing (scLRS) enhances tumor cell resolution by integrating methods for detecting splicing alterations and genetic variants. Schematic representation of how scLRS can visualize and combine multiple information types within the same cells to improve tumor cell resolution. Created in BioRender. Byrne, A. (2026) https://BioRender.com/mrmobm6 .

    Journal: Frontiers in Oncology

    Article Title: Beyond counting: how single-cell long-read sequencing turns transcriptome complexity into precision targets

    doi: 10.3389/fonc.2026.1800370

    Figure Lengend Snippet: Single-cell long-read sequencing (scLRS) enhances tumor cell resolution by integrating methods for detecting splicing alterations and genetic variants. Schematic representation of how scLRS can visualize and combine multiple information types within the same cells to improve tumor cell resolution. Created in BioRender. Byrne, A. (2026) https://BioRender.com/mrmobm6 .

    Article Snippet: Adopting third-generation long-read sequencing platforms, such as those developed by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), offers a solution to overcome this limitation and deepen our understanding of the transcriptome.

    Techniques: Single Cell, Sequencing