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MSRE-HTPrimer: a high-throughput and genome-wide primer design pipeline optimized for epigenetic research.

ABSTRACT: Methylation-sensitive restriction enzymes-polymerase chain reaction (MSRE-PCR) has been used in epigenetic research to identify genome-wide and gene-specific DNA methylation. Currently, epigenome-wide discovery studies provide many candidate regions for which the MSREqPCR approach can be very effective to confirm the findings. MSREqPCR provides high multiplexing capabilities also when starting with limited amount of DNA-like cfDNA to validate many targets in a time- and cost-effective manner. Multiplex design is challenging and cumbersome to define specific primers in an effective manner, and no suitable software tools are freely available for high-throughput primer design in a time-effective manner and to automatically annotate the resulting primers with known SNPs, CpG, repeats, and RefSeq genes. Therefore a robust, powerful, high-throughput, optimized, and methylation-specific primer design tool with great accuracy will be very useful.We have developed a novel pipeline, called MSRE-HTPrimer, to design MSRE-PCR and genomic PCR primers pairs in a very efficient manner and with high success rate. First, our pipeline designs all possible PCR primer pairs and oligos, followed by filtering for SNPs loci and repeat regions. Next, each primer pair is annotated with the number of cut sites in primers and amplicons, upstream and downstream genes, and CpG islands loci. Finally, MSRE-HTPrimer selects resulting primer pairs for all target sequences based on a custom quality matrix defined by the user. MSRE-HTPrimer produces a table for all resulting primer pairs as well as a custom track in GTF file format for each target sequence to visualize it in UCSC genome browser.MSRE-HTPrimer, based on Primer3, is a high-throughput pipeline and has no limitation on the number and size of target sequences for primer design and provides full flexibility to customize it for specific requirements. It is a standalone web-based pipeline, which is fully configured within a virtual machine and thus can be readily used without any configuration. We have experimentally validated primer pairs designed by our pipeline and shown a very high success rate of primer pairs: out of 190 primer pairs, 71 % could be successfully validated. The MSRE-HTPrimer software is freely available from http://sourceforge.net/p/msrehtprimer/wiki/Virtual_Machine/ as a virtual machine.

SUBMITTER: Pandey RV

PROVIDER: S-EPMC4779238 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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MSRE-HTPrimer: a high-throughput and genome-wide primer design pipeline optimized for epigenetic research.

Pandey Ram Vinay RV Pulverer Walter W Kallmeyer Rainer R Beikircher Gabriel G Pabinger Stephan S Kriegner Albert A Weinhäusel Andreas A

Clinical epigenetics 20160305

<h4>Background</h4>Methylation-sensitive restriction enzymes-polymerase chain reaction (MSRE-PCR) has been used in epigenetic research to identify genome-wide and gene-specific DNA methylation. Currently, epigenome-wide discovery studies provide many candidate regions for which the MSREqPCR approach can be very effective to confirm the findings. MSREqPCR provides high multiplexing capabilities also when starting with limited amount of DNA-like cfDNA to validate many targets in a time- and cost-e ...[more]

PMID: 26949424

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Project description:BackgroundIn some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this target gene approach are called "intron-flanking primers" or because they are located in exonic sequences which are usually conserved between related species, "conserved primers". They are useful for large-scale single nucleotide polymorphism (SNP) discovery and marker development, especially in species, such as wheat, for which a large number of ESTs are available but for which genome sequences and intron/exon boundaries are not available. To date, no suitable high-throughput tool is available for this purpose.ResultsWe have developed, the ConservedPrimers 2.0 pipeline, for designing intron-flanking primers for large-scale SNP discovery and marker development, and demonstrated its utility in wheat. This tool uses non-redundant wheat EST sequences, such as wheat contigs and singleton ESTs, and related genomic sequences, such as those of rice, as inputs. It aligns the ESTs to the genomic sequences to identify unique colinear exon blocks and predicts intron lengths. Intron-flanking primers are then designed based on the intron/exon information using the Primer3 core program or BatchPrimer3. Finally, a tab-delimited file containing intron-flanking primer pair sequences and their primer properties is generated for primer ordering and their PCR applications. Using this tool, 1,922 bin-mapped wheat ESTs (31.8% of the 6,045 in total) were found to have unique colinear exon blocks suitable for primer design and 1,821 primer pairs were designed from these single- or low-copy genes for PCR amplification and SNP discovery. With these primers and subsequently designed genome-specific primers, a total of 1,527 loci were found to contain one or more genome-specific SNPs.ConclusionThe ConservedPrimers 2.0 pipeline for designing intron-flanking primers was developed and its utility demonstrated. The tool can be used for SNP discovery, genetic variation assays and marker development for any target genome that has abundant ESTs and a related reference genome that has been fully sequenced. The ConservedPrimers 2.0 pipeline has been implemented as a command-line tool as well as a web application. Both versions are freely available at http://wheat.pw.usda.gov/demos/ConservedPrimers/.

Project description:BackgroundMicrosatellite (simple sequence repeat - SSR) and single nucleotide polymorphism (SNP) markers are two types of important genetic markers useful in genetic mapping and genotyping. Often, large-scale genomic research projects require high-throughput computer-assisted primer design. Numerous such web-based or standard-alone programs for PCR primer design are available but vary in quality and functionality. In particular, most programs lack batch primer design capability. Such a high-throughput software tool for designing SSR flanking primers and SNP genotyping primers is increasingly demanded.ResultsA new web primer design program, BatchPrimer3, is developed based on Primer3. BatchPrimer3 adopted the Primer3 core program as a major primer design engine to choose the best primer pairs. A new score-based primer picking module is incorporated into BatchPrimer3 and used to pick position-restricted primers. BatchPrimer3 v1.0 implements several types of primer designs including generic primers, SSR primers together with SSR detection, and SNP genotyping primers (including single-base extension primers, allele-specific primers, and tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing primers. DNA sequences in FASTA format can be batch read into the program. The basic information of input sequences, as a reference of parameter setting of primer design, can be obtained by pre-analysis of sequences. The input sequences can be pre-processed and masked to exclude and/or include specific regions, or set targets for different primer design purposes as in Primer3Web and primer3Plus. A tab-delimited or Excel-formatted primer output also greatly facilitates the subsequent primer-ordering process. Thousands of primers, including wheat conserved intron-flanking primers, wheat genome-specific SNP genotyping primers, and Brachypodium SSR flanking primers in several genome projects have been designed using the program and validated in several laboratories.ConclusionBatchPrimer3 is a comprehensive web primer design program to develop different types of primers in a high-throughput manner. Additional methods of primer design can be easily integrated into future versions of BatchPrimer3. The program with source code and thousands of PCR and sequencing primers designed for wheat and Brachypodium are accessible at http://wheat.pw.usda.gov/demos/BatchPrimer3/.

Project description:Circular RNA (circRNA) is a class of endogenous non-coding RNA characterized by a back-splice junction (BSJ). In general, large-scale circRNA BSJ detection is performed based on RNA sequencing data, followed by the selection and validation of circRNAs of interest using RT-qPCR with circRNA-specific PCR primers. Such a primer pair is convergent and functional on the circRNA template but divergent and non-functional on the linear host gene. Although a few circRNA primer design pipelines have been published, none of them offer large-scale, easy-to-use circRNA primer design. Other limitations are that these tools generally do not take into account assay specificity, secondary structures, and SNPs in the primer annealing regions. Furthermore, these tools are limited to circRNA primer design for humans (no other organisms possible), and no wet-lab validation is demonstrated. Here, we present CIRCprimerXL, a circRNA RT-qPCR assay design pipeline based on the primer design framework primerXL. CIRCprimerXL takes a circRNA BSJ position as input, and designs BSJ-spanning primers using Primer3. The user can choose to use the unspliced or spliced circRNA sequence as template. Prior to primer design, sequence regions with secondary structures and common SNPs are flagged. Next, the primers are filtered based on predicted specificity and the absence of secondary structures of the amplicon to select a suitable primer pair. Our tool is both available as a user-friendly web tool and as a stand-alone pipeline based on Docker and Nextflow, allowing users to run the pipeline on a wide range of computer infrastructures. The CIRCprimerXL Nextflow pipeline can be used to design circRNA primers for any species by providing the appropriate reference genome. The CIRCprimerXL web tool supports circRNA primer design for human, mouse, rat, zebrafish, Xenopus tropicalis, and C. elegans. The design process can easily be scaled up for the qPCR assay design of tens of thousands of circRNAs within a couple of hours. We show how CIRCprimerXL has been successfully used to design qPCR assays for over 15,000 human circRNAs of which 20 were empirically validated. CIRCprimerXL software, documentation, and test data can be found at: https://github.com/OncoRNALab/CIRCprimerXL. CIRCprimerXL is also implemented as a webtool at: https://circprimerxl.cmgg.be.

Dataset Information

MSRE-HTPrimer: a high-throughput and genome-wide primer design pipeline optimized for epigenetic research.

Publications

MSRE-HTPrimer: a high-throughput and genome-wide primer design pipeline optimized for epigenetic research.

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