The Experts below are selected from a list of 108 Experts worldwide ranked by ideXlab platform
Pengfeng Xiao - One of the best experts on this subject based on the ideXlab platform.
-
Probe optimization for Sequencing by Ligation.
Journal of biochemistry, 2014Co-Authors: Jing Chen, Xiaoting Qian, Pengfeng XiaoAbstract:Sequencing by Ligation (SBL) is a straightforward enzymatic method for interrogating DNA sequence, in which the Ligation efficiency and specificity of each probe play an essential role. Here, the number of labelled dyes in the probe, probe length and probe constituent were investigated to optimize the Ligation efficiency and specificity. First, the performance of double- and single-labelled fluorescent probes in SBL was evaluated. The experimental results showed that double-labelled fluorescent probes could yield a remarkable increase in the fluorescence intensities and avoid higher background compared with single-labelled fluorescent probes. Second, probes between 7- and 9-mers in length were designed to uniform Tm difference. We hoped the uniformed probes with smaller Tm difference could improve the Ligation efficiency. However, 8-mer probes with larger Tm difference showed stronger fluorescence intensities. Third, we evaluated whether probes containing deoxyinosines either in the 5' or the 3' end had influence on the Ligation efficiency. Consequently, probes containing deoxyinosines at the 5' termini might decrease the Ligation efficiency, and the accumulation of 3' terminal deoxyinosines in the Sequencing primers was likely to reduce the fluorescence intensity and the Ligation efficiency, which was inconsistent with the traditional viewpoint. The optimized probes will improve the Ligation efficiency and accuracy in SBL.
-
Sequencing-by-Ligation using oligonucleotide probes with 3'-thio-deoxyinosine.
Journal of biomedical nanotechnology, 2014Co-Authors: Jing Chen, Yunfei Bai, Hongmei Xie, Wenjie Wang, Pengfeng XiaoAbstract:We have developed a novel Sequencing-by-Ligation (SBL) system employing oligonucleotide probes containing 3'-thio-deoxyinosine on a microarray. The oligonucleotide probes were synthesized from 3'-S-(2-cyanoethyl-N,N-diisopropylphosphorothioamidite)-5'-O-(4,4' dimet-hoxytrityl) deoxyinosine. The resultant probes could be cleaved chemically by aqueous silver ions under mild conditions, generating a 5'-terminal phosphate group in a degradation oligodeoxynucleotide fragment. This 5'-terminal phosphate was used directly for detecting the corresponding bases in subsequent Sequencing cycles. The queried bases in the template were identified with eight cycles of Ligation and cleavage. The read length of our method could reach up to 40 bp with high accuracy. As this SBL method uses commercially available enzymes and standard microarrays, it will be amenable to automation and adaptation by the research community.
-
IWBBIO - Sequencing by Ligation with Double-Labeled Fluorescent Probes.
2014Co-Authors: Jing Chen, Pengfeng XiaoAbstract:We have synthesized a set of double-labeled fluorescent probes and evaluated the performance of these fluorescent probes in Sequencing by Ligation (SBL). The results indicated that the Ligation efficiency and specificity of dou- ble-labeled fluorescent probes was as higher as that of singe-labeled fluorescent probes. M oreover, d ouble-labeled f luorescent p robes y ielded a r emarkable i n- crease i n s ignal i ntensities co mpared t o s ingle-labeled f luorescent probes. I t could also reduce background and shorten exposition time compared to probes labeled with single FAM and FITC dyes. Thus, it could supplant singe-labeled fluorescent probes in SBL to increase Sequencing accuracy.
-
Sequencing by Ligation with double labeled fluorescent probes
IWBBIO, 2014Co-Authors: Jing Chen, Pengfeng XiaoAbstract:We have synthesized a set of double-labeled fluorescent probes and evaluated the performance of these fluorescent probes in Sequencing by Ligation (SBL). The results indicated that the Ligation efficiency and specificity of dou- ble-labeled fluorescent probes was as higher as that of singe-labeled fluorescent probes. M oreover, d ouble-labeled f luorescent p robes y ielded a r emarkable i n- crease i n s ignal i ntensities co mpared t o s ingle-labeled f luorescent probes. I t could also reduce background and shorten exposition time compared to probes labeled with single FAM and FITC dyes. Thus, it could supplant singe-labeled fluorescent probes in SBL to increase Sequencing accuracy.
-
endonuclease v assisted accurate cleavage of oligonucleotide probes controlled by deoxyinosine and deoxynucleoside phosphorothioate for Sequencing by Ligation
Analyst, 2012Co-Authors: Jing Tang, Zhiqiang Pan, Pengfeng XiaoAbstract:Sequencing-by-Ligation (SBL) is one of the next-generation Sequencing methods for massive parallel Sequencing. The ligated probes used in SBL should be accurately cleaved for a better Ligation in the next cycle. Here, a novel kind of oligonucleotide probe that could be accurately cleaved at the given position was proposed. Deoxynucleoside phosphorothioates were introduced into the deoxyoxanosine-containing oligonucleotide probes in order to increase the cleavage accuracy of endonuclease V on double-stranded DNA templates. The results illustrated that incorporating deoxynucleoside phosphorothioates could greatly reduce the effect of the nonsynchronous Sequencing primer, and the queried bases of the DNA templates were unambiguously identified with 5 cycles of Sequencing Ligations. Additionally, the read length can reach up to 25 bp with high accuracy. The SBL-based method is inexpensive, has high-throughput, and is easy to operate allowing massive scale-up, miniaturization and automation.
Jing Chen - One of the best experts on this subject based on the ideXlab platform.
-
Probe optimization for Sequencing by Ligation.
Journal of biochemistry, 2014Co-Authors: Jing Chen, Xiaoting Qian, Pengfeng XiaoAbstract:Sequencing by Ligation (SBL) is a straightforward enzymatic method for interrogating DNA sequence, in which the Ligation efficiency and specificity of each probe play an essential role. Here, the number of labelled dyes in the probe, probe length and probe constituent were investigated to optimize the Ligation efficiency and specificity. First, the performance of double- and single-labelled fluorescent probes in SBL was evaluated. The experimental results showed that double-labelled fluorescent probes could yield a remarkable increase in the fluorescence intensities and avoid higher background compared with single-labelled fluorescent probes. Second, probes between 7- and 9-mers in length were designed to uniform Tm difference. We hoped the uniformed probes with smaller Tm difference could improve the Ligation efficiency. However, 8-mer probes with larger Tm difference showed stronger fluorescence intensities. Third, we evaluated whether probes containing deoxyinosines either in the 5' or the 3' end had influence on the Ligation efficiency. Consequently, probes containing deoxyinosines at the 5' termini might decrease the Ligation efficiency, and the accumulation of 3' terminal deoxyinosines in the Sequencing primers was likely to reduce the fluorescence intensity and the Ligation efficiency, which was inconsistent with the traditional viewpoint. The optimized probes will improve the Ligation efficiency and accuracy in SBL.
-
Sequencing-by-Ligation using oligonucleotide probes with 3'-thio-deoxyinosine.
Journal of biomedical nanotechnology, 2014Co-Authors: Jing Chen, Yunfei Bai, Hongmei Xie, Wenjie Wang, Pengfeng XiaoAbstract:We have developed a novel Sequencing-by-Ligation (SBL) system employing oligonucleotide probes containing 3'-thio-deoxyinosine on a microarray. The oligonucleotide probes were synthesized from 3'-S-(2-cyanoethyl-N,N-diisopropylphosphorothioamidite)-5'-O-(4,4' dimet-hoxytrityl) deoxyinosine. The resultant probes could be cleaved chemically by aqueous silver ions under mild conditions, generating a 5'-terminal phosphate group in a degradation oligodeoxynucleotide fragment. This 5'-terminal phosphate was used directly for detecting the corresponding bases in subsequent Sequencing cycles. The queried bases in the template were identified with eight cycles of Ligation and cleavage. The read length of our method could reach up to 40 bp with high accuracy. As this SBL method uses commercially available enzymes and standard microarrays, it will be amenable to automation and adaptation by the research community.
-
IWBBIO - Sequencing by Ligation with Double-Labeled Fluorescent Probes.
2014Co-Authors: Jing Chen, Pengfeng XiaoAbstract:We have synthesized a set of double-labeled fluorescent probes and evaluated the performance of these fluorescent probes in Sequencing by Ligation (SBL). The results indicated that the Ligation efficiency and specificity of dou- ble-labeled fluorescent probes was as higher as that of singe-labeled fluorescent probes. M oreover, d ouble-labeled f luorescent p robes y ielded a r emarkable i n- crease i n s ignal i ntensities co mpared t o s ingle-labeled f luorescent probes. I t could also reduce background and shorten exposition time compared to probes labeled with single FAM and FITC dyes. Thus, it could supplant singe-labeled fluorescent probes in SBL to increase Sequencing accuracy.
-
Sequencing by Ligation with double labeled fluorescent probes
IWBBIO, 2014Co-Authors: Jing Chen, Pengfeng XiaoAbstract:We have synthesized a set of double-labeled fluorescent probes and evaluated the performance of these fluorescent probes in Sequencing by Ligation (SBL). The results indicated that the Ligation efficiency and specificity of dou- ble-labeled fluorescent probes was as higher as that of singe-labeled fluorescent probes. M oreover, d ouble-labeled f luorescent p robes y ielded a r emarkable i n- crease i n s ignal i ntensities co mpared t o s ingle-labeled f luorescent probes. I t could also reduce background and shorten exposition time compared to probes labeled with single FAM and FITC dyes. Thus, it could supplant singe-labeled fluorescent probes in SBL to increase Sequencing accuracy.
Kuo-ping Chiu - One of the best experts on this subject based on the ideXlab platform.
-
Palindromic sequence impedes Sequencing-by-Ligation mechanism.
BMC systems biology, 2012Co-Authors: Yu-feng Huang, Sheng-chung Chen, Yih-shien Chiang, Tzu-han Chen, Kuo-ping ChiuAbstract:Current next-generation Sequencing (NGS) platforms adopt two types of Sequencing mechanisms: by synthesis or by Ligation. The former is employed by 454 and Solexa systems, while the latter by SOLiD system. Although the pros and cons for each Sequencing mechanism have more or less been discussed in a number of occasions, the potential obstacle imposed by palindromic sequences has not yet been addressed. To test the effect of the palindromic region on Sequencing efficacy, we clonally amplified a paired-end ditag sequence composed of a 24-bp palindromic sequence flanked by a pair of tags from the E. coli genome. We used the near homogeneous fragments produced from MmeI digestion of the amplified clone to generate a Sequencing library for SOLiD 5500xl sequencer. Results showed that, traditional ABI sequencers, which adopt Sequencing-by-synthesis mechanism, were able to read through the palindromic region. However, SOLiD 5500xl was unable to do so. Instead, the palindromic region was read as miscellaneous random sequences. Moreover, readable tag sequence turned obscure ~2 bp prior to the palindromic region. Taken together, we demonstrate that SOLiD machines, which employ Sequencing-by-Ligation mechanism, are unable to read through the palindromic region. On the other hand, Sequencing-by-synthesis sequencers had no difficulty in doing so.
-
palindromic sequence impedes Sequencing by Ligation mechanism
BMC Systems Biology, 2012Co-Authors: Yu-feng Huang, Sheng-chung Chen, Kuo-ping Chiu, Yih-shien Chiang, Tzu-han ChenAbstract:Background Current next-generation Sequencing (NGS) platforms adopt two types of Sequencing mechanisms: by synthesis or by Ligation. The former is employed by 454 and Solexa systems, while the latter by SOLiD system. Although the pros and cons for each Sequencing mechanism have more or less been discussed in a number of occasions, the potential obstacle imposed by palindromic sequences has not yet been addressed.
-
Palindromic sequence impedes Sequencing-by-Ligation mechanism
BMC Systems Biology, 2012Co-Authors: Yu-feng Huang, Sheng-chung Chen, Yih-shien Chiang, Tzu-han Chen, Kuo-ping ChiuAbstract:Background Current next-generation Sequencing (NGS) platforms adopt two types of Sequencing mechanisms: by synthesis or by Ligation. The former is employed by 454 and Solexa systems, while the latter by SOLiD system. Although the pros and cons for each Sequencing mechanism have more or less been discussed in a number of occasions, the potential obstacle imposed by palindromic sequences has not yet been addressed. Methods To test the effect of the palindromic region on Sequencing efficacy, we clonally amplified a paired-end ditag sequence composed of a 24-bp palindromic sequence flanked by a pair of tags from the E. coli genome. We used the near homogeneous fragments produced from Mme I digestion of the amplified clone to generate a Sequencing library for SOLiD 5500xl sequencer. Results Results showed that, traditional ABI sequencers, which adopt Sequencing-by-synthesis mechanism, were able to read through the palindromic region. However, SOLiD 5500xl was unable to do so. Instead, the palindromic region was read as miscellaneous random sequences. Moreover, readable tag sequence turned obscure ~2 bp prior to the palindromic region. Conclusions Taken together, we demonstrate that SOLiD machines, which employ Sequencing-by-Ligation mechanism, are unable to read through the palindromic region. On the other hand, Sequencing-by-synthesis sequencers had no difficulty in doing so.
Zhiqiang Pan - One of the best experts on this subject based on the ideXlab platform.
-
endonuclease v assisted accurate cleavage of oligonucleotide probes controlled by deoxyinosine and deoxynucleoside phosphorothioate for Sequencing by Ligation
Analyst, 2012Co-Authors: Jing Tang, Zhiqiang Pan, Pengfeng XiaoAbstract:Sequencing-by-Ligation (SBL) is one of the next-generation Sequencing methods for massive parallel Sequencing. The ligated probes used in SBL should be accurately cleaved for a better Ligation in the next cycle. Here, a novel kind of oligonucleotide probe that could be accurately cleaved at the given position was proposed. Deoxynucleoside phosphorothioates were introduced into the deoxyoxanosine-containing oligonucleotide probes in order to increase the cleavage accuracy of endonuclease V on double-stranded DNA templates. The results illustrated that incorporating deoxynucleoside phosphorothioates could greatly reduce the effect of the nonsynchronous Sequencing primer, and the queried bases of the DNA templates were unambiguously identified with 5 cycles of Sequencing Ligations. Additionally, the read length can reach up to 25 bp with high accuracy. The SBL-based method is inexpensive, has high-throughput, and is easy to operate allowing massive scale-up, miniaturization and automation.
-
Single nucleotide polymorphism genotyping and point mutation detection by Ligation on microarrays.
Journal of nanoscience and nanotechnology, 2011Co-Authors: Jing Tang, Pengfeng Xiao, Zhiqiang Pan, Dongrui Zhou, Min PanAbstract:Based on the principle of Sequencing by Ligation, a novel method referred to as "MiniSequencing by Ligation" for detecting known single nucleotide variant in high-throughput assay formats is reported in this article. by designing a Sequencing primer and fluorescently labeled probes each with a "discriminating" base according to the single base variation, this method can be used to analyze single nucleotide polymorphism (SNP) or point mutation in a number of samples in parallel. In our current study, three known nucleotides at a given position of three DNA templates with different CG contents are firstly genotyped on aldehyde-modified slide to testify the feasibility and optimize the reaction conditions. Then, by performing Ligation reaction between phosphorylated 5' end of the Sequencing primer and hydroxylated 3' end of the labeled probe or phosphorylated 5' end of the labeled probe and hydroxylated 3' end of the Sequencing primer, a SNP locus (rs1800497 (C/T)) and a point mutation (Mt1555 (A/G)) have been accurately detected on aldehyde-modified microarray and Sepharose beads in acrylamide gel, respectively. It has demonstrated that "miniSequencing by Ligation," as a promising methodology, can perform point mutation and SNP analysis in a simple, cost-effective, robust and high-throughput way.
Yu-feng Huang - One of the best experts on this subject based on the ideXlab platform.
-
Palindromic sequence impedes Sequencing-by-Ligation mechanism.
BMC systems biology, 2012Co-Authors: Yu-feng Huang, Sheng-chung Chen, Yih-shien Chiang, Tzu-han Chen, Kuo-ping ChiuAbstract:Current next-generation Sequencing (NGS) platforms adopt two types of Sequencing mechanisms: by synthesis or by Ligation. The former is employed by 454 and Solexa systems, while the latter by SOLiD system. Although the pros and cons for each Sequencing mechanism have more or less been discussed in a number of occasions, the potential obstacle imposed by palindromic sequences has not yet been addressed. To test the effect of the palindromic region on Sequencing efficacy, we clonally amplified a paired-end ditag sequence composed of a 24-bp palindromic sequence flanked by a pair of tags from the E. coli genome. We used the near homogeneous fragments produced from MmeI digestion of the amplified clone to generate a Sequencing library for SOLiD 5500xl sequencer. Results showed that, traditional ABI sequencers, which adopt Sequencing-by-synthesis mechanism, were able to read through the palindromic region. However, SOLiD 5500xl was unable to do so. Instead, the palindromic region was read as miscellaneous random sequences. Moreover, readable tag sequence turned obscure ~2 bp prior to the palindromic region. Taken together, we demonstrate that SOLiD machines, which employ Sequencing-by-Ligation mechanism, are unable to read through the palindromic region. On the other hand, Sequencing-by-synthesis sequencers had no difficulty in doing so.
-
palindromic sequence impedes Sequencing by Ligation mechanism
BMC Systems Biology, 2012Co-Authors: Yu-feng Huang, Sheng-chung Chen, Kuo-ping Chiu, Yih-shien Chiang, Tzu-han ChenAbstract:Background Current next-generation Sequencing (NGS) platforms adopt two types of Sequencing mechanisms: by synthesis or by Ligation. The former is employed by 454 and Solexa systems, while the latter by SOLiD system. Although the pros and cons for each Sequencing mechanism have more or less been discussed in a number of occasions, the potential obstacle imposed by palindromic sequences has not yet been addressed.
-
Palindromic sequence impedes Sequencing-by-Ligation mechanism
BMC Systems Biology, 2012Co-Authors: Yu-feng Huang, Sheng-chung Chen, Yih-shien Chiang, Tzu-han Chen, Kuo-ping ChiuAbstract:Background Current next-generation Sequencing (NGS) platforms adopt two types of Sequencing mechanisms: by synthesis or by Ligation. The former is employed by 454 and Solexa systems, while the latter by SOLiD system. Although the pros and cons for each Sequencing mechanism have more or less been discussed in a number of occasions, the potential obstacle imposed by palindromic sequences has not yet been addressed. Methods To test the effect of the palindromic region on Sequencing efficacy, we clonally amplified a paired-end ditag sequence composed of a 24-bp palindromic sequence flanked by a pair of tags from the E. coli genome. We used the near homogeneous fragments produced from Mme I digestion of the amplified clone to generate a Sequencing library for SOLiD 5500xl sequencer. Results Results showed that, traditional ABI sequencers, which adopt Sequencing-by-synthesis mechanism, were able to read through the palindromic region. However, SOLiD 5500xl was unable to do so. Instead, the palindromic region was read as miscellaneous random sequences. Moreover, readable tag sequence turned obscure ~2 bp prior to the palindromic region. Conclusions Taken together, we demonstrate that SOLiD machines, which employ Sequencing-by-Ligation mechanism, are unable to read through the palindromic region. On the other hand, Sequencing-by-synthesis sequencers had no difficulty in doing so.
