Dear all,
according to your experiences what is a better approach for non-model plant RNA-seq - refererence based assembly or de novo assembly? I will do RNA seq experiment in Papaver somniferum (opium poppy) and wondering if would be better to do reference based assembly using Arabidopsis thaliana genome or to do de novo assembly using Trinity tool. I am interested mainly in difference gene expression analysis and in subsequent annotation of some new drought response genes.
Thank you for your opinions
Martin
Hi Martin
I would recommend a reference guided assembly only if you can find a genome of a closely related species. Then by mapping the reads on the genome and by looking at the percentage of mapped reads (both uniquely and multiple mapping) you could have an idea about how much are close the two species.
In the past we used a combination of reference guided and de novo assembly, in which we used a reference guided approach with the reads mapping on a closely related species and performing a de novo assembly on the unmapped reads to be sure that we did not lose important transcripts which might not be conserved.
I have looked on NCBI and I saw that there are two Papaveracea genomes available (https://www.ncbi.nlm.nih.gov/genome/?term=txid3465[Organism:exp]), maybe you could have a look and check if they are similar enough.
Have a nice day
Riccardo
Bioinformatics data analyst at www.sequentiabiotech.com
Hi Martin,
I would say you're much better off with a de novo assembly. Trinity is very good for de novo transcriptome assembly, but combining multiple assemblies may give you a more accurate representation of the true transcripts. Check out this paper:
Article Combining independent de novo assemblies optimizes the codin...
Good luck!
Kyle
Hi Martin
I would recommend a reference guided assembly only if you can find a genome of a closely related species. Then by mapping the reads on the genome and by looking at the percentage of mapped reads (both uniquely and multiple mapping) you could have an idea about how much are close the two species.
In the past we used a combination of reference guided and de novo assembly, in which we used a reference guided approach with the reads mapping on a closely related species and performing a de novo assembly on the unmapped reads to be sure that we did not lose important transcripts which might not be conserved.
I have looked on NCBI and I saw that there are two Papaveracea genomes available (https://www.ncbi.nlm.nih.gov/genome/?term=txid3465[Organism:exp]), maybe you could have a look and check if they are similar enough.
Have a nice day
Riccardo
Bioinformatics data analyst at www.sequentiabiotech.com
Hello,
@Kyle, thank you for great paper, combining of more de novo assembly programs sounds like a good idea. Currently, the only way to do this by "user-friendly" way is probably through Galaxy server (https://usegalaxy.org/?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fnate%2Ftrinity_psc%2Ftrinity_psc%2F0.0.1&version=0.0.1&__identifer=wvp15dw8du)
because I have not access to other efficient computational resources.
@Riccardo, thank you for your answer, but I was unable to find these two Papaveracea genomes and your link seems not to be functional. But in case that Papaver genome was sequenced, it would be a great help for me, could you check the link you send me previously? Thanks in advance
Martin
Dear Martin
this is the link to the genomes I found on NCBI: https://www.ncbi.nlm.nih.gov/genome/?term=txid3465[Organism:exp]
I have the feeling that Research Gate might be blocking it, maybe you could try to copy and paste it in Google Chrome/Safari/Firefox instead of clicking directly on it. If the link still does not work you could go to NCBI and search within the "Genome" database the following word: txid3465[Organism:exp]
I hope this will work. By this way I found the genome assemblies of Eschscholzia californica and Macleaya cordata.
Please let me know if you need more help.
Kind regards
Riccardo Aiese Cigliano
Bioinformatics data analyst at www.sequentiabiotech.com
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