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Avni B. Santani - One of the best experts on this subject based on the ideXlab platform.
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Mung Bean Nuclease treatment abolished the high molecular DNA smear in RainDance captured DNA after end repair.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:A. An overview of the workflow. Aliquots of 100ng of DNA enriched through microdroplet-PCR were either treated with Mung-Bean Nuclease or untreated as a control. The differentially treated aliquots were end-repaired, concatenated, processed into TruSeq libraries and sequenced on MiSeq in parallel to rule out any batch effect. B. Electrophoresis analysis of DNA samples on a high-sensitivity DNA chip using the Agilent 2100 Bioanalyzer. DNA samples in all 4 lanes were derived from 200pg of the same batch of captured DNA. From left to right, the lanes are, “L” lane-the DNA size ladder, lane 1-untreated DNA enriched by RainDance microdroplet-PCR prior to end repair, lane 2- untreated DNA enriched by RainDance microdroplet-PCR post end repair, lane 3- Mung Bean Nuclease treated DNA enriched by RainDance microdroplet-PCR prior to end repair, lane 4- Mung Bean Nuclease treated DNA enriched by RainDance microdroplet-PCR post end repair.
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Comparison of variants detected for the same sample either treated or not treated with Mung Bean Nuclease.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:*: Variants detected only when samples were treated with Mung Bean Nuclease.
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On-target efficiency for libraries prepared from samples either treated or untreated with the Mung Bean Nuclease.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:On-target efficiency for libraries prepared from samples either treated or untreated with the Mung Bean Nuclease.
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Mung Bean Nuclease treatment significantly increases on-target efficiency for DNA enriched through RainDance microdroplet-PCR.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:Aliquots of RainDance enriched DNA for the same sample were either treated or not treated with Mung Bean Nuclease, processed into TruSeq libraries, and sequenced on MiSeq as illustrated in Figure 1A. Plotted is the mean value of on-target efficiency of 3 samples that went through parallel treatments (also see Table 2). Nuclease treatment leads to significantly higher on-target efficiency (*p = 0.018, one-tail paired t test; error bar, SEM).
Kajia Cao - One of the best experts on this subject based on the ideXlab platform.
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Mung Bean Nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.
PLoS ONE, 2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni SantaniAbstract:Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a Nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that Nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.
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Mung Bean Nuclease treatment abolished the high molecular DNA smear in RainDance captured DNA after end repair.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:A. An overview of the workflow. Aliquots of 100ng of DNA enriched through microdroplet-PCR were either treated with Mung-Bean Nuclease or untreated as a control. The differentially treated aliquots were end-repaired, concatenated, processed into TruSeq libraries and sequenced on MiSeq in parallel to rule out any batch effect. B. Electrophoresis analysis of DNA samples on a high-sensitivity DNA chip using the Agilent 2100 Bioanalyzer. DNA samples in all 4 lanes were derived from 200pg of the same batch of captured DNA. From left to right, the lanes are, “L” lane-the DNA size ladder, lane 1-untreated DNA enriched by RainDance microdroplet-PCR prior to end repair, lane 2- untreated DNA enriched by RainDance microdroplet-PCR post end repair, lane 3- Mung Bean Nuclease treated DNA enriched by RainDance microdroplet-PCR prior to end repair, lane 4- Mung Bean Nuclease treated DNA enriched by RainDance microdroplet-PCR post end repair.
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Comparison of variants detected for the same sample either treated or not treated with Mung Bean Nuclease.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:*: Variants detected only when samples were treated with Mung Bean Nuclease.
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On-target efficiency for libraries prepared from samples either treated or untreated with the Mung Bean Nuclease.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:On-target efficiency for libraries prepared from samples either treated or untreated with the Mung Bean Nuclease.
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Mung Bean Nuclease treatment significantly increases on-target efficiency for DNA enriched through RainDance microdroplet-PCR.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:Aliquots of RainDance enriched DNA for the same sample were either treated or not treated with Mung Bean Nuclease, processed into TruSeq libraries, and sequenced on MiSeq as illustrated in Figure 1A. Plotted is the mean value of on-target efficiency of 3 samples that went through parallel treatments (also see Table 2). Nuclease treatment leads to significantly higher on-target efficiency (*p = 0.018, one-tail paired t test; error bar, SEM).
Catherine A. Stolle - One of the best experts on this subject based on the ideXlab platform.
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Mung Bean Nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.
PLoS ONE, 2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni SantaniAbstract:Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a Nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that Nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.
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Mung Bean Nuclease treatment abolished the high molecular DNA smear in RainDance captured DNA after end repair.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:A. An overview of the workflow. Aliquots of 100ng of DNA enriched through microdroplet-PCR were either treated with Mung-Bean Nuclease or untreated as a control. The differentially treated aliquots were end-repaired, concatenated, processed into TruSeq libraries and sequenced on MiSeq in parallel to rule out any batch effect. B. Electrophoresis analysis of DNA samples on a high-sensitivity DNA chip using the Agilent 2100 Bioanalyzer. DNA samples in all 4 lanes were derived from 200pg of the same batch of captured DNA. From left to right, the lanes are, “L” lane-the DNA size ladder, lane 1-untreated DNA enriched by RainDance microdroplet-PCR prior to end repair, lane 2- untreated DNA enriched by RainDance microdroplet-PCR post end repair, lane 3- Mung Bean Nuclease treated DNA enriched by RainDance microdroplet-PCR prior to end repair, lane 4- Mung Bean Nuclease treated DNA enriched by RainDance microdroplet-PCR post end repair.
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Comparison of variants detected for the same sample either treated or not treated with Mung Bean Nuclease.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:*: Variants detected only when samples were treated with Mung Bean Nuclease.
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On-target efficiency for libraries prepared from samples either treated or untreated with the Mung Bean Nuclease.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:On-target efficiency for libraries prepared from samples either treated or untreated with the Mung Bean Nuclease.
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Mung Bean Nuclease treatment significantly increases on-target efficiency for DNA enriched through RainDance microdroplet-PCR.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:Aliquots of RainDance enriched DNA for the same sample were either treated or not treated with Mung Bean Nuclease, processed into TruSeq libraries, and sequenced on MiSeq as illustrated in Figure 1A. Plotted is the mean value of on-target efficiency of 3 samples that went through parallel treatments (also see Table 2). Nuclease treatment leads to significantly higher on-target efficiency (*p = 0.018, one-tail paired t test; error bar, SEM).
Tanya Tischler - One of the best experts on this subject based on the ideXlab platform.
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Mung Bean Nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.
PLoS ONE, 2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni SantaniAbstract:Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a Nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that Nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.
-
Mung Bean Nuclease treatment abolished the high molecular DNA smear in RainDance captured DNA after end repair.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:A. An overview of the workflow. Aliquots of 100ng of DNA enriched through microdroplet-PCR were either treated with Mung-Bean Nuclease or untreated as a control. The differentially treated aliquots were end-repaired, concatenated, processed into TruSeq libraries and sequenced on MiSeq in parallel to rule out any batch effect. B. Electrophoresis analysis of DNA samples on a high-sensitivity DNA chip using the Agilent 2100 Bioanalyzer. DNA samples in all 4 lanes were derived from 200pg of the same batch of captured DNA. From left to right, the lanes are, “L” lane-the DNA size ladder, lane 1-untreated DNA enriched by RainDance microdroplet-PCR prior to end repair, lane 2- untreated DNA enriched by RainDance microdroplet-PCR post end repair, lane 3- Mung Bean Nuclease treated DNA enriched by RainDance microdroplet-PCR prior to end repair, lane 4- Mung Bean Nuclease treated DNA enriched by RainDance microdroplet-PCR post end repair.
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Comparison of variants detected for the same sample either treated or not treated with Mung Bean Nuclease.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:*: Variants detected only when samples were treated with Mung Bean Nuclease.
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On-target efficiency for libraries prepared from samples either treated or untreated with the Mung Bean Nuclease.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:On-target efficiency for libraries prepared from samples either treated or untreated with the Mung Bean Nuclease.
-
Mung Bean Nuclease treatment significantly increases on-target efficiency for DNA enriched through RainDance microdroplet-PCR.
2014Co-Authors: Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. SantaniAbstract:Aliquots of RainDance enriched DNA for the same sample were either treated or not treated with Mung Bean Nuclease, processed into TruSeq libraries, and sequenced on MiSeq as illustrated in Figure 1A. Plotted is the mean value of on-target efficiency of 3 samples that went through parallel treatments (also see Table 2). Nuclease treatment leads to significantly higher on-target efficiency (*p = 0.018, one-tail paired t test; error bar, SEM).
Douglas G Inglis - One of the best experts on this subject based on the ideXlab platform.
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characterization of mucosa associated bacterial communities of the mouse intestine by terminal restriction fragment length polymorphism utility of sampling strategies and methods to reduce single stranded dna artifacts
Journal of Microbiological Methods, 2009Co-Authors: Estela Costa, Nathan J Puhl, Brent L Selinger, Douglas G InglisAbstract:Terminal restriction fragment length polymorphism (T-RFLP) is a molecular technique used for comparative analysis of microbial community structure and dynamics. We evaluated three sampling methods for recovering bacterial community DNA associated with intestinal mucosa of mice (i.e. mechanical agitation with PBS, hand washing with PBS containing Tween 80, and direct DNA extraction from mucosal plugs). In addition, the utility of two methods (i.e. Klenow fragment and Mung-Bean Nuclease) to reduce single-stranded DNA artifacts was tested. T-RFLP analysis indicated that diverse communities of bacteria are associated with mucosa of the ileum, cecum, and descending colon of mice. Although there was no significant difference in bacterial community structure between the mechanical agitation and direct DNA extraction methods regardless of intestinal location, community diversity was reduced for the hand wash method in the colon. The use of Klenow fragment and Mung-Bean Nuclease have been reported to eliminate single-stranded DNA artifacts (i.e. pseudo-T-restriction fragments), but neither method was beneficial for characterizing mucosa-associated bacterial communities of the mouse cecum. Our study showed that the mechanical agitation and direct plug extraction methods yielded equivalent bacterial community DNA from the mucosa of the small and large intestines of mice, but the latter method was superior for logistical reasons. We also applied a combination of different statistical approaches to analyze T-RFLP data, including statistical detection of true peaks, analysis of variance for peak number, and group significance test, which provided a quantitative improvement for the interpretation of the T-RFLP data.
