[maker-devel] strand of single exon EST from fasta

[Jacques Dainat]

Hello,

I’m annotating several strains of a same fungus, and I have stranded RNAseq for all of them. I’m using MAKER3.
Let’s say I’m annotating the species1 using its species-specific assembled transcripts that are in gff. I know that MAKER cannot do anything about the strand coming from the est_gff. In order to check that everything went fine during my transcriptome assembly and the strands correctly defined, I checked the annotation within a browser. I can see the strands from my transcripts in gff format were perfect (match with the proteins strands / and with abinitio prediction strands / and ORFs are OK).

As I wanted to take advantage on my other strains RNAseq I decided to use them within this annotation. As the transcriptome assemblies of these RNAseq have been done based on their corresponding genomes, I cannot use the gff files. Indeed, the location are not corresponding to the  genome of my species1. So I decided to extract the sequences in fasta format to feed MAKER with (alt_est parameter).
When I visualise those transcript alignements I was really surprised by the strands decided by MAKER. It seems completely random, while all the est fasta sequences from a same locus are given in the same strand.

So, I have two questions:
1) How the strand is decided for single exon EST provided in fasta format ? (I thought it was based on the longest ORF)
2) Is it normal that the second annotation using these alt_est is worse (far less gene models) than the previous one ? (I thought the strand of my single exon alt_ests would not play a role during the the annotation process. Or maybe it’s another biais from these alt_est => loci less well defined ?)



Here 3 examples: The top green track has the correct strand and is based on the gff file. The bottom green cluster tracks are fasta sequences from the other strains aligned through MAKER. (I dont’t know if it could play a role but all sequences from a same locus have been sent to MAKER in the same strand). 


Thank you very much for your help,

Jacques Dainat
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