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

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.

CRISPR loci-PCR as Tool for Tracking Azospirillum sp. Strain B510

Azospirillum-based plant and soil inoculants are widely used in agriculture. The inoculated Azospirillum strains are commonly tracked by both culture-dependent and culture-independent methods, which are time-consuming or expensive. In this context, clustered regularly interspaced short palindromic repeats (CRISPR) loci structure is unique in the bacterial genome, including some Azospirillum species. Here, we investigated the use of CRISPR loci to track specific Azospirillum strains in soils systems by PCR. Primer sets for Azospirillum sp. strain B510 were designed and evaluated by colony and endpoint PCR. The CRISPRloci-PCR approach was standardized for Azospirillum sp. strain B510, and its specificity was observed by testing against 9 different Azospirillum strains, and 38 strains of diverse bacterial genera isolated from wheat plants. The CRISPRloci-PCR approach was validated in assays with substrate and wheat seedlings. Azospirillum sp. strain B510 was detected after of two weeks of inoculation in both sterile and nonsterile substrates as well as rhizosphere grown in sterile substrate. The CRISPRloci-PCR approach was found to be a useful molecular tool for specific tracking of Azospirillum at the strain level. This technique can be easily adapted to other microbial inoculants carrying CRISPR loci and can be used to complement other microbiological techniques.

Evaluating and Improving Small Subunit rRNA PCR Primer Coverage for Bacteria, Archaea, and Eukaryotes Using Metagenomes from Global Ocean Surveys

PCR amplification and sequencing of marker genes are a low-cost technique for monitoring prokaryotic and eukaryotic microbial communities across space and time but will work optimally only if environmental organisms match PCR primer sequences exactly. In this study, we evaluated how well primers match globally distributed short-read oceanic metagenomes.

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

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.