scholarly journals Minimum Multicolored Subgraph Problem in Multiplex PCR Primer Set Selection and Population Haplotyping

2006 ◽  
pp. 758-766 ◽  
Author(s):  
M. T. Hajiaghayi ◽  
K. Jain ◽  
L. C. Lau ◽  
I. I. Măndoiu ◽  
A. Russell ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Pcr Primer

scholarly journals Development of Multiplex PCR Primer Sets for the Identification of Rice Varieties

2005 ◽  
Vol 7 (2) ◽  
pp. 87-94 ◽  
Author(s):  
Kazunori Shinmura ◽  
Hiroshi Kanagawa ◽  
Takashi Mikami ◽  
Takeshi Fukumori
Keyword(s):  
Multiplex Pcr ◽  
Rice Varieties ◽  
Primer Sets ◽  
Pcr Primer

Algorithms for Multiplex PCR Primer Set Selection with Amplification Length Constraints

2007 ◽  
pp. 241-258 ◽  
Author(s):  
K. M. Konwar ◽  
I. I. Mǎndoiu ◽  
A. C. Russell ◽  
A. A. Shvartsman
Keyword(s):  
Multiplex Pcr ◽  
Pcr Primer

scholarly journals Designing Highly Multiplex PCR Primer Sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)

2021 ◽  
Author(s):  
David Zhang ◽  
Nina Xie ◽  
Michael Wang ◽  
Ping Song ◽  
Yifan Wang ◽  
...  
Keyword(s):  
Simulated Annealing ◽  
Multiplex Pcr ◽  
Dna Sequences ◽  
Simulated Annealing Algorithm ◽  
Likelihood Estimation ◽  
Stochastic Algorithm ◽  
Gene Fusions ◽  
Dimer Formation ◽  
Primer Sets ◽  
Pcr Primer

Abstract The design of highly multiplex PCR primers to amplify and enrich many different DNA sequences is increasing in biomedical importance as new mutations and pathogens are identified. One major challenge in the design of highly multiplex PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of highly multiplex PCR primer sets that minimize primer dimer formation. Our approach uses a rapidly computable Loss function to approximate the degree of primer dimer formation within a primer set, and randomly swaps primers in the set with alternative candidates using a simulated annealing algorithm. In a 96-plex PCR primer set (192 primers), we show that we can reduce the fraction of primer dimers from 90.7% in a naively designed PCR primer set to 4.9% in our optimized primer set. Running the optimized 96-plex primer set on FFPE DNA samples from cancer patients, we likewise observe a low fraction of primer dimer reads. Even when scaling to 384-plex (768 primers), the PCR primer set designed by our algorithm maintains low primer dimer fraction. In addition to NGS, we also show that our SADDLE-designed multiplex primer sets can be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.

Feasiblity and Implication of Bidirectional Sequencing Using a Multiplex Framework 2 Region Primer for Somatic Mutation Analysis of the Immunoglobulin (IgH) Heavy Chain in Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4706-4706
Author(s):  
Adin Nelson ◽  
Ivelise Rijo ◽  
Zhigang Zhang ◽  
Andrew D. Zelenetz ◽  
Ariela Noy
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Multiplex Pcr ◽  
Somatic Mutation ◽  
Mutation Rate ◽  
Heavy Chain ◽  
Consensus Sequence ◽  
Lymphocytic Leukemia ◽  
Cdr3 Region ◽  
Reverse Sequence ◽  
Pcr Primer

Abstract Background: A somatic mutation rate of >2% of the immunoglobulin (IgH) heavy chain gene, in comparison to germline DNA, is a positive prognostic indicator in chronic lymphocytic leukemia (CLL). Genomic DNA is amplified using the Biomed-2 multiplex PCR primer set, sequenced in the reverse direction only using a 3′ JH primer, and compared to the germline sequence in VBase, a comprehensive directory of all human germline variable region sequences compiled from over a thousand published sequences. It is unknown whether forward sequencing using the multiplex 5′ Biomed-2 primers in a single reaction is feasible or whether doing so improves the accuracy of the mutational analysis. Methods: DNA was extracted from peripheral blood or bone marrow mononuclear cells from patients with CLL using the QIAGEN QIAspin DNA mini-kit. The FR2 region of the IgH gene was amplified using tube B of the Biomed-2 multiplex PCR primer set, separated in 2% agarose gel, excised, and prepared for sequencing using the QIAGEN QIAquick gel extraction kit. Bidirectional DNA sequencing was performed with the full set of multiplex 5′ FR2 primers combined in one reaction for the forward sequence and in a second reaction with the Biomed-2 3′ JH primer for the reverse sequence. A consensus sequence was obtained and mutation rates were calculated based on the consensus sequence and the reverse sequence separately. Results: The FR2 region was amplified from 30 CLL patients. 20 samples produced bidirectional consensus DNA sequences, 2 sequenced only with multiplex FR2 primers, 6 sequenced only with a JH primer, and 2 produced no sequences. Compared with unidirectional JH sequencing, bidirectional sequencing did not reassign any patient in the germline IgH group to the mutated category. In 15 patients, the uni- and bi-directional sequencing were identical. In 4 patients, a single basepair difference was noted. A random permutation test yields a two-sided p-value as 12.5%, indicating that no significant difference was detected between the uni- and bi-directional sequencing. Multiplex bidirectional sequencing did provide sequence information within the CDR3 region at the 3′ terminus of the amplimer which is partially truncated when using the JH primer alone. Conclusion: Bidirectional sequencing does not provide a somatic IgH mutation rate that differs significantly from that obtained from the reverse sequence alone and does not likely influence the IgH mutational prognostic assignment. Nonetheless, the bidirectional consensus sequence adds bases in the CDR3 region which can be used for research applications such as the generation of patient-specific primers for PCR. It is also noteworthy that multiplex PCR using the Biomed-2 primers is feasible.

scholarly journals The web-based multiplex PCR primer design software Ultiplex and the associated experimental workflow: up to 100- plex multiplicity

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jie Yuan ◽  
Ji Yi ◽  
Meixiao Zhan ◽  
Qingqing Xie ◽  
Ting Ting Zhen ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Secondary Structures ◽  
Primer Design ◽  
Design Tool ◽  
Web Based ◽  
Tumour Diagnosis ◽  
Design Software ◽  
Nonspecific Amplification ◽  
User Friendly ◽  
Pcr Primer

Abstract Background A large number of variants have been employed in various medical applications, such as providing medication instructions, disease susceptibility testing, paternity testing, and tumour diagnosis. A high multiplicity PCR will outperform other technologies because of its lower cost, reaction time and sample consumption. To conduct a multiplex PCR with higher than 100 plex multiplicity, primers need to be carefully designed to avoid the formation of secondary structures and nonspecific amplification between primers, templates and products. Thus, a user-friendly, highly automated and highly user-defined web-based multiplex PCR primer design software is needed to minimize the work of primer design and experimental verification. Results Ultiplex was developed as a free online multiplex primer design tool with a user-friendly web-based interface (http://ultiplex.igenebook.cn). To evaluate the performance of Ultiplex, 294 out of 295 (99.7%) target primers were successfully designed. A total of 275 targets produced qualified primers after primer filtration, and 271 of those targets were successfully clustered into one compatible PCR group and could be covered by 108 primers. The designed primer group stably detected the rs28934573(C > T) mutation at lower than a 0.25% mutation rate in a series of samples with different ratios of HCT-15 and HaCaT cell line DNA. Conclusion Ultiplex is a web-based multiplex PCR primer tool that has several functions, including batch design and compatibility checking for the exclusion of mutual secondary structures and mutual false alignments across the whole genome. It offers flexible arguments for users to define their own references, primer Tm values, product lengths, plex numbers and tag oligos. With its user-friendly reports and web-based interface, Ultiplex will provide assistance for biological applications and research involving genomic variants.

scholarly journals Development of multiplex PCR primer sets for identification of major strawberry (Fragaria×ananassa Duchesne) cultivars in Japan

2008 ◽  
Vol 10 (3) ◽  
pp. 111-115
Author(s):  
Kimihisa Tasaki ◽  
Yuuki Kashiwaya ◽  
Shun-ichi Kobayashi ◽  
Masayuki Amagai
Keyword(s):  
Multiplex Pcr ◽  
Primer Sets ◽  
Pcr Primer
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