Multiple sequence alignment of all retrieved sequences was performed using ClustalX [30] to identify and remove duplicate, splice variant, reading frame shift, truncated or otherwise non usable MS4A sequences from the dataset.
An analysis of known duplicated and alternatively spliced genes in Drosophila suggests that the use of this method cannot fully exclude misclassification, but that the frequency of false calls should overall be low (see Methods).
Briefly, the script was fed Newbler assemblies in ACE format and performed the following steps: (i) identify broken reads based on read names, (ii) construct graph using Pearl Graph Module [ 96], (iii) identify CC and BCC using Pearl Graph Module function, (iv) flag each BCC as putatively allelic, duplicated, or alternative splicing according to the criteria described below.
If the duplicated gene has multiple splice variants with different binding interactions and knockouts of different splice sites between the two duplicates occur, then the result would be subfunctionalization of the duplicates with the ultimate retention of both.
We confirmed lower frequency of alternative splicing in duplicated genes compared to non-duplicated ones.
In considering transcriptome k -mers, two cases arise that are particularly problematic for pseudogenes: processed pseudogenes with integrated splice junctions and duplicated pseudogenes that may have highly similar splice junctions to their parent genes.
We also found these duplicated genes having alternative splicing were under tighter evolutionary constraints compared to those having no alternative splicing, and had an enrichment of genes that participate in molecular transducer activities.
We actually found a tendency to increase in proportion of alternative splicing in duplicated genes in both zebrafish and fly, even though they are not statistically significant.
As you would expect from alleles of the same gene, all the contigs from merged components (highly variable alleles of the same gene) were more likely to match the same gene in NCBI-NR than were contigs from alternatively spliced or duplicated gene components.
The Reverse Transcription Polymerase Chain Reaction (RT-PCR) showed a very clear band in D. melanogaster and T. castaneum adult cDNA, as in A. mellifera with constitutive exon 3. Direct sequencing of these amplification products confirmed that these duplicated exons were alternatively spliced.
