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unexpectedly low depth of coverage after mapping with MiniMap2 #1089

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sjfleck opened this issue Jul 20, 2023 · 3 comments
Open

unexpectedly low depth of coverage after mapping with MiniMap2 #1089

sjfleck opened this issue Jul 20, 2023 · 3 comments

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@sjfleck
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sjfleck commented Jul 20, 2023

Hello, I have 3 samples I'm working with. By comparing the total ONT sequenced read length and the estimated haploid genome sizes
sample 1: 24.96x coverage
sample 2: 22.36x coverage
sample 3: 22.47x coverage

after assembling the genomes, reducing them to haploid assemblies with Purge Haplotigs, and mapping the reads back on to the haploid assemblies using minimap2, I calculated the read coverage directly:
sample 1: 22.79x coverage
sample 2: 21.20x coverage
sample 3: 21.16x coverage

This all seems to agree with my initial calculations. The strange part is, when I create histograms of the coverage, sample 2 has one peak at 12x coverage. It had this peak before and after Purge Haplotigs and I'm not sure why it's so low. Sample 1 and 3 had 2 peaks originally- one at ~11 and another at ~22. After Purge Haplotigs, Sample 1 and 3 only had the haploid peak at ~22.

sample_peaks

Can you help me understand what could have happened or simply why sample 2 had such a low coverage peak? Thank you for any guidance you can provide
@lh3
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lh3 commented Jul 20, 2023

This seems strange. Do these samples come from same or similar species? What is the total assembly sizes before and after purge dup?

@sjfleck
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sjfleck commented Jul 20, 2023 •
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These are 3 separate species from the same genus. Sample 1 and 3 are closely related, but not sister species. Sample 2 is in the same genus, but in a different section. For the assembly sizes, these are before Purge Haplotigs:
sample 1: 1,076 MB
sample 2: 796 MB
sample 3: 1,082 MB

And these are the assembly sizes after Purge Haplotigs:
sample 1: 852 MB
sample 2: 776 MB
sample 3: 842 MB

These assembly lengths are close to the estimated haploid genome size calculated from flow cytometry:
sample 1: 875 MB
sample 2: 829 MB
sample 3: 869 MB

@lh3
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lh3 commented Jul 20, 2023

I don't know... What does the k-mer histogram of hifiasm log look like?

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@lh3 @sjfleck