Transcriptome assembly for polyploid species

[DarioC]

Dear seqanswers.com community,

I am working with a hexaploid organism. No genome is sequence is available yet. De novo assembly of 454 reads resulted in contigs that are often chimera of reads belonging to homeologous genomes. I was wondering if anyone has experience with transcriptome assembly using EST datasets as reference. I have plenty of ESTs available and coming these from clones are genome specific. However, reads will allign to multiple redundant ESTs.

Any suggestions or ideas?

Thanks, Dario

[colindaven]

Hi Dario

Sounds tricky.

A first step might be to cluster your EST collection with cd-hit to get rid of the redundant ESTs ?

For de novo assembly perhaps you can tighten up the alignment parameters to avoid chimeras? What percentage of contigs are chimeric ?

[DarioC]

Thanks for the cd-hit suggestion. I never used it, I will give it a try. Roughly I would say that 10% of the sequences may be chimera of homeologous genes. There is no certain way to differentiate them from 'true' contigs. We are going to add depth with Illumina sequencing. Thanks. Dario

[baochengguo]

Hi Dario,

How is your project going on? I am also working on polyploid transcriptomes right now. I am trying with different parameters in newblerm. But it is difficult to construct and distinguish paralogs. Do you have any comments? Thanks.

[DarioC]

Hi Dario,

How is your project going on? I am also working on polyploid transcriptomes right now. I am trying with different parameters in newblerm. But it is difficult to construct and distinguish paralogs. Do you have any comments? Thanks.

Hi baochengguo:

For that project, we simplified the polyploid reference (454) by grouping transcript variants/homeologs/chimera/close paralogs into groups (isogroups). We mapped the reads (illumina) on the contigs and taken the sum as the expression level for the group. If you are interested you can find more details in our paper: http://www.biomedcentral.com/1471-2164/12/492

This was not the most elegant solution, but good enough for this first part of the study. We are now trying to do assisted assembly of illumina reads using very stringent mapping on wheat ESTs. This will allow assembly of genome specific contigs, but we are still sequencing...

[baochengguo]

Hi baochengguo:

For that project, we simplified the polyploid reference (454) by grouping transcript variants/homeologs/chimera/close paralogs into groups (isogroups). We mapped the reads (illumina) on the contigs and taken the sum as the expression level for the group. If you are interested you can find more details in our paper: http://www.biomedcentral.com/1471-2164/12/492

This was not the most elegant solution, but good enough for this first part of the study. We are now trying to do assisted assembly of illumina reads using very stringent mapping on wheat ESTs. This will allow assembly of genome specific contigs, but we are still sequencing...

Hi Dario,

Thanks so much for the reply. I have downloaded your paper and will read it. If any question, I will bother you again.

[spenthil]

This paper might also be of interest: Meta-IDBA: a de Novo assembler for metagenomic data. "We introduce the Meta-IDBA algorithm for assembling reads in metagenomic data, which contain multiple genomes from different species."
http://bioinformatics.oxfordjournals...27/13/i94.full