A comparison of junction-primer designing tools
Balagannavar Govindkumar, Basavaraju Kavyashree and Patel Krishna (*Correspondence: Acharya KK, kshitish@ibab.ac.in) |
|
Feature |
RExPrimer |
Primer Blast |
QuantPrime |
PRIMERGENS |
RJPrimers |
BatchPrimer3 (2008) |
MRPrimerW2 (2019) |
ExPrimer |
Ex-Ex Primer |
Status* |
Not working |
Working |
commercial |
Not Working |
Not working |
not working |
Working |
Discontinued |
Working |
Input |
HUGO gene name, NCBI gene ID, NCBI gene symbol, chromosome [band] |
Accession, GI or sequence in FASTA format |
Transcript ID, or user can enter the transcript sequence in FASTA format which will be searched using BLAST and identifier will be retrieved |
User can design different types of primers such as primers covering CpG island, primers around TSS, primers around restriction enzyme cut-site region or normal primer. User can upload sequence in fasta format to design normal and primers around restriction enzyme cut-site region. To design primers covering CpG island and around TSS, user can provide list of Human gene symbols. User can further select if he wants to design sequence specific primer, fragment specific primer, probe specific primer, or probe. Click here for more... |
Upload or paste the sequence in fasta format. Since it is Transposable elements based PCR pimer design program, 17 different repeat databases are available for selection which includes databases of a dedicated species and database of different eukaryotic species |
paste sequence and upload fasta file |
NCBI Gene Symbol, GeneBank Accission, NCBI Gene ID, Aliases, keyword |
Upload gene genbank file, transcript ID, exon number for creating junction |
NCBI gene symbol, paste the gene Genbank file, upload the gene genbank file |
Organism |
Human |
No option |
All |
Human, mouse, yeast, zebrafish, drosophila, C. Elegan, Guinea Pig, Anopheles gambiae |
No dedicated option. |
No DB data connected |
Human, Mouse, Rat, Zebrafish, Cow, Pig, Thale cress, Fruit fly, C. elegans |
No option |
Human, Mouse, Rat |
Options |
Primers only |
Primers only |
Primers only |
Primers and probes |
Primers only |
Primers only |
Primers, TaqMan probes, primers avoiding SNP (only for human) |
Primers |
Primers and probes |
Information given for the select genes |
Official symbol, gene type (protein-coding or non protein coding), full name of the gene, position: chr(nn)[band], size of gene, contig accession, mRNA accession, protein accession, orientation, gene ID |
None |
None |
None |
None |
None |
Gene Symbol,RefSeq ID, Gene ID, Aliases, Keyword |
None |
Official Gene Symbol, Gene ID, RefSeq ID , Ensembl gene ID |
alternative splice varients of a gene displayed |
NA |
No |
NA |
NA |
NA |
None |
No |
None |
Yes, all the forms present in NCBI database (like NM_ and XM_) |
Transcript information given in the initial pages |
mRNA accession, transcript structure |
None |
None |
None |
None |
None |
No |
None |
length, exon/intron sequence and flanking region information for every exon/intron of all the transcript, mRNA accession, transcript structure |
Graphical display of all transcripts of a gene |
NA |
No |
NA |
NA |
NA |
No |
No |
None |
Yes, interactive and mouse over will provide additional information of the exons |
Junctional primer selection |
Not primary utility, and not a user friendly feature |
Not the primary utility; and not a user friendly feature. However, junction primers can be designed using option "Primer must span an exon-exon junction" for field "Exon junction span" |
Not primary utility; and not a user friendly feature. However, junction primers can be designed using option "End-Point sqPCR (accept splice variant)" for field "End-point sqPCR (accept splice variant hits)" Click here for more... |
Not primary utility, and not a user friendly feature |
Primary utility of software is to be able to design TE-based unique repeat junction primer. So there are various types of junction primers possible. For instance, Repeat junction marker (RJM), Repeat junction-junction marker (RJJM), insertion-site-based polymorphism (ISBP), inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-based insertion polymorphism (RBIP) Click here for more... |
yes |
Not allowed (its automated) |
Not user friendly |
Primary utility & user friendly |
Option for specifying E-E junction(s)** |
Not available |
Not available |
Not available |
None |
Not applicable |
None |
No |
Not available |
Yes. User can enter the serial number of exons to create junction(s). User can create junction from non consecutive exons as well (new, hypothetical junctions can be explored) |
One/Both primers as junction primers |
One |
One |
One |
NA |
One |
None |
one |
One or both |
One or both |
Obtaining a pair where one primer is E-E junction while the other is from an intron |
No |
No |
No |
None |
Not applicable |
No |
No |
None |
Yes |
Primer repeat library |
Human, Rodent, Drosophila |
Human, Rodent, Arabidopsis, Fruitfly, Rice, Mammals, Fungi, C Elegans, A Gambiae, Zebrafish |
None |
None |
Not applicable |
No |
No |
None |
None |
Primer specificity |
In-silico PCR |
BLAST |
BLAST |
BLAST |
None |
No |
No |
BLAST |
BLAST |
SNP validation |
1. Validated SNPs, 2. All SNPs |
Yes |
None |
None |
None |
Yes |
Yes (only for Human) |
None |
None |
Options for users to choose Tm methods |
None |
Santa Lucia 1998(Default), Breslauer et. al. 1986Also salt correction methods: Owczarzy et. al. 2009, Santa Lucia 1998, Schildkraut and Lifson 1965 |
None |
Santa Lucia 1998(Default), Breslauer et al. 1986, and Rychlik et al. 1990 |
None |
None |
No |
Theoretical, arbitrary |
Santa Lucia 1998 (Default), Breslauer et. al. 1986, Salt Concentration, Marmur and Doty, Howley et. al., Arbitrary |
Result Page |
Primer detail: Sequence, position, size, Tm, GC%, primer stabilityProduct details: Sequence, size, position, Chr(xx) |
Transcript info: ID, name, size, orientation, transcript structureTranscript structure: Length, orientation of each exons; link to FASTA and Genbank view, and BLAST GenomicProtein details: Detail of the protein translated from the submitted transcript, its accession ID, length, orientation, and CCDS. linkPrimer detail: Sequence, length, start position, stop position, Tm, GC%, self complimentary, self 3' complementaryProduct details: Sequence, sizeExon Junction: End position of exon n^start position of exon n+1 Click here for more... |
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Transcript info: Transcript ID, name of the gene, EC no.Primer details: Sequence, length, Tm, GC%Product details: size, Tm, GC%, optimal annealing temperature |
Transcript info:NonePrimer details:Sequence, start position,length. Tm, GC%Product size:Product size |
a main HTML page containing the primer design summary of all input sequences; an HTML table page listing all designed primers and primer properties (see example); a tab-delimited text file with the same contents in the HTML table page; and a detailed primer view page for each sequence with successfully designed primers (see example). A simple click on the links on the main HTML page or HTML table page will display the primer view. The primer list can be directly saved as a text file or an Excel file for further editing or primer ordering. Click here for more... |
Gene name, RefSeq ID, Tm, Primers, product length, penalty score |
Result can be viewd in 2 different formats and can be downloaded in 3 different formats. Junction location, type and its source is also displayed.Primer details: Sequencesm start and end position, length, Tm, GC%, pair annealing and self annealing score, insertion type.Product details: Product size |
Primer details: Sequence, position, size, Tm, GC%, stability (score), Option: more details, Product sequence, Show other primer(s) , Product as well as submitted sequence Product details: Sequence, size, position In case of junction primers: Spliced sequence information Click here for more... |
URL |
click to visit |
click to visit |
click to visit |
click to visit |
click to visit |
click to visit |
click to visit |
NA |
click to visit |
Experimental validation |
Yes |
No |
Yes |
paper not available |
Yes |
Yes |
No |
NA |
Yes |
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