Comments (4)
mschatz
commented on August 13, 2024
Hi, Thanks for your interest. This is a fairly poor fit - can you send the
histogram file or link to the genomescope webpage with your results?
Thanks!
Mike
…On Fri, Jun 9, 2023 at 3:26 AM Sally Lau ***@***.***> wrote:
Hi there
I am having trouble fitting genomescope2 to my HiFi data and I am hoping
if you could point me to the right direction on how to proceed.
The genome size estimation is what I expected for my species. The linear
plot is showing the full model overlapping with errors, but the
transformed_linear_plot is much clearer in presenting the data. So I can
see that this is a diploid with high heterozygosity (which makes sense as
this is a wild marine invertebrate specimen). But for some reason
genomescope isn't indicating the data is characterised by high
heterozygosity, and that the full model is only showing 1 peak. I wonder
what might be causing this issue? Thank you!
[image: linear_plot]
<https://user-images.githubusercontent.com/60337082/244620824-d95aaae1-5b1a-4bc8-9199-20e9a14c4458.png>
[image: transformed_linear_plot]
<https://user-images.githubusercontent.com/60337082/244620856-8369274c-70b7-4c71-80fc-40efd9aeeed5.png>
property min max
Homozygous (aa) 98.5315% 100%
Heterozygous (ab) 0% 1.46848%
Genome Haploid Length 1,671,603,854 bp 3,366,896,208 bp
Genome Repeat Length 878,309,778 bp 1,769,066,190 bp
Genome Unique Length 793,294,076 bp 1,597,830,018 bp
Model Fit 46.958% 92.6345%
Read Error Rate 0.0826566% 0.0826566%
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sallycylau
commented on August 13, 2024
Hi Mike
Thanks so much for the quick reply. Here is the genomescope link (http://genomescope.org/genomescope2.0/analysis.php?code=tC8i3MBprfe3k57Irbbz). .hist file was generated with meryl. Any tips for how to fit the model would be much appreciated. Thank you!
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mschatz
commented on August 13, 2024
Hi Sally,
I was able to get a slightly better fit here:
http://qb.cshl.edu/genomescope/genomescope2.0/analysis.php?code=T9COTpC3AJlsDduOF6rw
The only change was to provide a hint on the "Average k-mer coverage for
polyploid genome" which I set to 40x. This gives much higher heterozygosity
(3.04%) although the genomesize is cut in half (this is expected because I
effectively changed which peaks are the homozygous and heterozygous
values). Do you know if 3% heterozygosity and this genome size is
realistic? The fit is still relatively poor and doesnt do a great job
capturing all of the kmers with coverage around 20x. Is this from a single
individual or where multiple samples sequenced together? Else you might
have a bit of contamination present - have you run an assembly yet? Have
you tried screening it with kraken?
Good luck!
Mike
…On Mon, Jun 12, 2023 at 3:31 AM Sally Lau ***@***.***> wrote:
Hi Mike
Thanks so much for the quick reply. Here is the genomescope link (
http://genomescope.org/genomescope2.0/analysis.php?code=tC8i3MBprfe3k57Irbbz).
.hist file was generated with meryl. Any tips for how to fit the model
would be much appreciated. Thank you!
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sallycylau
commented on August 13, 2024
Hi Mike
Thanks for your tips and sorry for the late follow up. I had to drop this project for a while and am now catching back up!
In your supplied link... I think setting "Average k-mer coverage for polyploid genome" to 40x which resulted in 3% het and 1.3 GB genome actually makes some sense....
I went ahead and assembled the genome with pretty aggressive purging (hifiasm -l3 + purged_dup) as this was found to give me the best completed assemblies (judging by N50 and contig #, and Complete BUSCO didn't vary much by changing hifiasm -l. Purged_dups definitely needed for duplications). I ended with a primary assembly of 1.8GB with BUSCO eukaryota C:96.90 [S: 91.80, D:5.10], F:1.2, M:1.9; and an alternate assembly of 1.3GB.
I ran Kraken on the purged assembles and 98.89% came back unclassified for primary and 99.97% for alternate, so I think contamination isn't the main issue here? We definitely only sequenced a single individual. This is a wild Antarctic brittle star from a place that receives a lot of current and gene flow from other places.... not ideal I am aware!
I ran smudgeplot as well and it does show this species as a diploid (but very heterozygous)
I tried to run Genomescope again with "Average k-mer coverage for polyploid genome" set as 30x which the estimated genome size came back as 1.9GB which matches the value in my purged primary assembly. However the fit is still quite poor. http://genomescope.org/genomescope2.0/analysis.php?code=PAA93V8MApgPGmKlPZrh
I wonder if there is still any way I could improve my Genomescope results? Thank you!
Best regards
Sally
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Related Issues (20)
- FAQ not up to date regarding ploidy HOT 1
- No file uploaded error message HOT 3
- Low model fit while ran Genomescope 2.0
- Strange genomescope result HOT 1
- High heterozygous? HOT 3
- No file upload message HOT 1
- Unable to converge HOT 3
- 503 Service Unavailable HOT 2
- mergeing HiFi data of two samples didn't increase hetorozygosity
- Heterozygosity rate < 0 HOT 3
- expectations for pooled samples HOT 2
- Heterozygous tetraploid genome model fit HOT 2
- GenomeScope Output from PacBio Hifi ccs Reads is Confounding HOT 3
- Model and observations don't converge HOT 3
- dup (in figure) and bias (in model.txt) HOT 2
- Follow up RE: Confounding GenomeScope Output HOT 5
- Ploidy determination HOT 1
- Heterozygous peak identified as errors? HOT 3
- Estimated genome size is half HOT 2
- Should a larger K value be chosen? HOT 1
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