how to deal with many CARs

[shuiyanwu778]

Dear Agora group, I am reconstructing ancestral gene orders using the AGORA software. The main challenge I am facing is that I have identified hundreds of CARs with decreasing numbers of genes. The number of genes in adjacent CARs is very similar, making it difficult to infer the true ancestral chromosome number. Are there any methods or parameters that I can adjust to improve this situation? My data consists of eight species from the same family, with chromosome numbers ranging from 21 to 23. For each genome, I used only the longest transcript to identify orthologs. In total, I used approximately 12,000 single-copy orthologs identified by orthoFinder for the AGORA analysis. I have included the gene numbers for the first 34 CARs below, and hope that you could help me with this problem. Thank you very much!

1 CAR_1 394 2 CAR_2 302 3 CAR_3 294 4 CAR_4 284 5 CAR_5 267 6 CAR_6 262 7 CAR_7 260 8 CAR_8 248 9 CAR_9 235 10 CAR_10 230 11 CAR_11 224 12 CAR_12 219 13 CAR_13 215 14 CAR_14 193 15 CAR_15 183 16 CAR_17 175 17 CAR_16 175 18 CAR_18 172 19 CAR_19 170 20 CAR_20 166 21 CAR_21 164 22 CAR_22 150 23 CAR_23 147 24 CAR_24 146 25 CAR_25 138 26 CAR_26 131 27 CAR_27 123 28 CAR_29 109 29 CAR_28 109 30 CAR_30 106 31 CAR_33 105 32 CAR_32 105 33 CAR_31 105 34 CAR_34 102

Kind regards, Cyan

[alouis72]

Hi Cyan, What workflow have you tried? agora-basic? agora-vertebrate? agora-plants?

Are the genomes well assembled (chromosome level)?

Do you have the possibility to add an outgroup to the studied species? Regards, Alex

[shuiyanwu778]

Hi Alex， Thanks for the quick reply. I used agora-vertebrate, and all genomes are chr-level. About outgroup, do you mean I’ d better use N1 rather than N0 as the ancestor to draw karyotype or is there any other parameters in Agora to do this? Indeed, I found N0 is always more fragmented than N1, but the number of CARS in N1 is still too much to deduce the real ancestral chr number for my data. Hope that you could help me with this problem, and feel free to contact me if anything unclear.

Best, Cyan

[alouis72]

Hi Cyan, The number of CARs inferred by AGORA will depend on many variables related to your data. The reconstruction will depend on the number of extant genomes you provide, the phylogenetic distance between species, the quality of genome annotations, the specific structure of each genome, and the quality of the orthologous assignments you performed.

AGORA is not the tool to “infer the true ancestral chromosome number.” The algorithm will do its best to reconstruct an ancestral gene order but it is not its purpose to infer the exact number of ancestral chromosomes.

For example: As shown in the publication (Figure 4), even though zoofish studies infer 12 chromosomes for the Poaceae ancestor in grasses, AGORA infers 19 larger CARs (and more), which can be mapped to the 12 chromosomes of rice (used as a proxy for the ancestral grass karyotype). The links between scaffolds cannot be inferred by the algorithm, likely due to the lack of conserved synteny between species in centromeric regions.

However, for your data, you may want to try the “agora-generic” script to attempt scaffolding in order to improve the size of the CARs. Results, however, should be carefully analyzed to check for potential over-assembly.

I hope this helps.

best regards, Alex

[shuiyanwu778]

Hi Alex, Thank you very much for the quick reply. I have tried "Agora-generic", and it shows a similar result. Do you mean that AGORA could not infer the exact ancestral number for any datasets and we have to modify the result manually? My main trouble is that for my ten chromosome-level genomes within the same family, I have constructed their genomic synteny using NGenomeSyn software and knew that there is only one to two chromosome-level fusions/fissions between species. These ten geomes have more than 12000 single-copy orthologs and now I want to contruct their ancestor karytype using AGORA and investigate the factual gene order rearragements among species. However, AGORA seems hard to infer the exact number of ancestral chromosomes even for these closely related species with similar chromosome numbers. So I am not sure if I must infer the ancestral chromosome level manually, or is there any other parameters in AGORA could help me with this question.

Cheers, Cyan