Human Splicing Finder
Version 2.4

Ensembl database release:	44 (April 2007)
Automatically select the longest transcript:	Yes No
Search for SNPs related to the analyzed sequence:	Yes No
Display ESE/ESS:	All Do not display ESE/ESS located at splice junctions
Number of nucleotides surrounding the exon
Both 5' and 3' introns (max: 1000):
Or
5' intron (max: 1000):
3' intron (max: 1000):

Choose a sequence by

-----------------[Select]----------------- Gene Name (e.g. DMD) Ensembl Transcript ID (e.g. ENST00000357033) Ensembl Gene ID (e.g. ENSG00000198947) Consensus CDS (e.g. CCDS14233) RefSeq Peptide ID (e.g. NP_004000) Pasting your own sequence : [Optional - Exon number: ][Optional - Intron number: ]

Paste your reference sequence into the following box ( characters remaining, plain text only):

Paste your mutant sequence into the following box ( characters remaining, plain text only):

You can highlight a specific section of your sequence and thus defining an exon. For this, please specify the first and last nucleotide positions:

• First nucleotide
• Last nucleotide

Paste your mutant list into the follow box:

(e.g. c.4250T>A) For more information on how to fill in this form, please refer to the help located at the bottom of this page.

Paste your sequence into the following box ( characters remaining, plain text only):

You must enter a 100 bp sequence.

Paste your identifiers into the following box:

(e.g. ENST00000358776///c.303+3A>G) For more information on how to fill in this form, please refer to the help located at the bottom of this page.

Advanced parameters (Show/Hide)

You can select all or a subset of matrices (Show/Hide) and set their thresholds to display Splicing Elements.

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Select an analysis type

Analyze a sequence

Either enter your own sequence or select a given gene and find splicing elements. This mode allows you to perform a complete analysis on a single sequence. It can be followed by the visualization of the impact of a mutation (substitution, insertion, deletion) or the impact study of SNPs related to this sequence.

Analyze mutation(s)

Visualize the impact of a given mutation on splicing elements of your own sequences or a selected transcript from the database (Ensembl 44). This mode allows you to either perform a complete analysis of several mutants against a transcript stored in our database (batch analysis) or compare two sequences.

Each mutant must be described at the cDNA level according to the mutation nomenclature. This application currently supports:

• substitutions - c.10019G>A
• insertions - c.1542_1543insA
• deletions - c.2230_2231del
• duplications - c.5434_5437dup
• indels
• inversions

Mutants must be separated by either a space character or a breakline character. The system will automatically and independantly for each mutant check for the proper exon and introns.

This mode can be followed by a SNP search or a "quick mutant" analysis on either the reference or mutant sequences or both.

Branch point analysis

Enter your own sequence and find potential branching points. This mode can be followed by a SNP search or a "quick mutant" analysis.

Splice site analysis

Either enter your own sequence or select a given gene and find potential acceptor and donor sites for splicing. This mode allows you to perform an analysis on a sequence to find potential donor and acceptor splice sites. It can be followed by a SNP search.

Multiple transcript analysis

Enter an Ensembl transcript ID followed by three slashes and a mutant written according to the international mutation nomenclature (at the cDNA level). Different mutants must be separated by either a space character or a breakline character.

Choose a sequence

Gene name

Please note that if you have difficulties finding a gene using a gene symbol, you can check the HUGO Gene Nomenclature Committee for its proper symbol.

Ensembl transcript ID

Select a transcript corresponding to its Ensembl transcript identifier.

Ensembl gene ID

Select a gene corresponding to its Ensembl gene identifier.

Consensus CDS

Select a transcript corresponding to its Consensus CDS. NCBI and Ensembl share the same sequence for this transcript.

RefSeq Peptide ID

Retrieve in Ensembl's database a gene/transcript corresponding to the NCBI RefSeq Peptide identifier.

Paste your own sequence

Instead of using our database, you may wish to paste our own sequences. Before analyzing them, the system will clean your sequences and remove all characters that aren't A, C, G or T.

Options

Automatically select the longest transcript

If you are sure that you must perform an analysis on the longest transcript of a given gene, this option will force the system to choose the longest transcript for you. Please note that if at least two transcripts match the requirement of being the longest, a screen will be displayed for you to choose the correct one. This option doesn't work if you use a Ensembl Transcript ID as a sequence identifier. Default value: Yes.

Single Nucleotide Polymorphisms

Human Splicing Finder is able to check on the Ensembl variation database for SNPs related to the analyzed sequence. Default value: No.

Nucleotides surrounding the exon

You can decide to display intronic nucleotides surrounding the exon. By default HSF will use a value of 0. Intronic nucleotides are limited to 1000 at 5' and 3' ends of the exon. If you type a value superior to 1000, the system will automatically change it to 1000. Default value: 0.

Advanced parameters

These parameters only work for mutants and multiple transcript analysis.

• Process sequences: To save computation time, HSF will only process the region where the mutation is located. To view all elements on the whole sequence, please use the "Full sequence" option. Default: Only variants.
• Add images: To speed up calculation, images are disabled by default. You can enable them with this option. Default: No.
• Specific matrix for position +3: You can use a new matrix to detect potentially pathogenous splicing mutants in position +3. Default value: No.

Matrices and thresholds

You can use all or a selection of matrices listed in this field. Furthermore, you can adjust threshold values by entering the desirated values in the appropriate boxes. Thresholds range from 0 to 100 in all matrices used in HSF. Available matrices are listed in the "Select matrices" section.