Definition

'5'_ORF_Extender' is a tool that can perform a systematic identification of extended coding regions (CDS) at the 5´ end of known mRNAs, using an EST-based approach.


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First, the user is guided to import RefSeq mRNA genomic coordinates and sequences from UCSC genome browser.
In addition, a table, matching each mRNA or EST sequence of the investigated organism to a genomic locus, is imported following its obtainment from UniGene data.

At the end of this step, the software will have automatically determined which EST is candidate for extension of its cognate mRNA 5´ CDS, due to the greater extension of the EST sequence on the genome in comparison with the position of mRNA 5´ end:






Database construction - Homo sapiens

Firstly, the human RefSeq flat file (version October 18, 2011) was downloaded from the UCSC (University of California, Santa Cruz) Genome Bioinformatics web site (http://genome.ucsc.edu/ - "Tables" section). The text file was imported into the appropriate 5'_ORF_Extender database table (following the software user guide) to obtain a local RefSeq database with 31,903 entries containing human known reference mRNA sequences ("NM_" prefix, thus excluding RefSeq entries not supported by experimental evidence, such as "XM_" models), corresponding to 18,665 distinct loci (a locus can have more than one mRNA isoform registered in RefSeq). It is possible to only select and further analyze mRNA entries without an in-frame stop codon upstream of the described initiation codon, which are thus candidates for a possible extension at 5´ end, because the presence of such a stop codon would indicate that the 5´ UTR sequence cannot be part of a longer continuous CDS. This also implies that a database of all RefSeq mRNAs that are bona fide complete at the 5´ end of their CDS is therefore generated.
     The genome alignment data for human ESTs - assigned by UniGene (see section 2.3 below) to the same locus of the mRNAs candidates for a possible extension at 5´ end - were then downloaded from the UCSC site (7,166,113 entries, version October 19, 2011) and imported into the appropriate 5'_ORF_Extender table. Each human mRNA (without an in-frame stop codon upstream of the described initiation codon) was then compared with all the human EST assigned to the same locus by analyzing the coordinates of the pre-computed genome alignments for mRNAs and ESTs obtained by UCSC. Only those EST sequence entries presenting additional nucleotides upstream of the known 5´ mRNA end and therefore candidate to potentially extend the mRNA CDS at its 5´, were downloaded (159,378 candidates).
    The whole analysis for H. sapiens, including data import and processing, required about 3 days for completion (1 additional day was required to obtain the updated UniGene table from 'UniGene Tabulator' and a further half day to import it prior to starting the analysis).

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