<p>They just want to know the ploidy of the sample. In a diploid sample, e.g. humans, there are two haplotypes. This is mostly important for haplotype resolution (i.e. get the two full genomes in a diploid).</p>
<p>Note that elsewhere in the brochure that you link it writes:</p>
<blockquote>
<p>Fast and efficient assembly of even
the largest genomes with haplotype
resolution of complex polyploids</p>
</blockquote>
<p>If you are working with a diploid species, that translates to 20-30X coverage. That seems low to me for the older tech of CLR/Sequel sequencing, but they are the experts.</p>
<p>Different recommendations posted <a href="https://pacbio.gs.washington.edu/" rel="nofollow noreferrer">here</a> by a service provider suggest 40-50X for structural variant analysis, which seems more reasonable.</p>

CC BY-SA 4.0
2021-09-09T21:25:29.587Z
There are still situations where I might not know the ploidity due to a lack of data. At least for now I was not able to find any data on either phylum: Cnidaria or  the class: Scyphozoa or the order: Rhizostomeae. Or in the case of *Sequoia sempervirens* how did they estimate the ploidity in advance?  Thanks for the insights!
ilam engl
CC BY-SA 4.0
2021-09-09T22:06:28.450Z
@ilamengl I think that there are some methods you can look into: 1) karyotype analysis, 2) computational tools, here is one example: https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-018-2128-z
Maximilian Press
CC BY-SA 4.0
2021-09-09T22:13:17.720Z
Additionally, there is a ploidy database which does appear to have cnidarians: https://cromanpa94.github.io/ACC/
Maximilian Press
CC BY-SA 4.0
2021-09-09T20:52:56.500Z
2021-09-09T20:52:56.500Z
Maximilian Press
<p>Unless you want to reconstruct all haplotypes, you can sequence to ~30X regardless of ploidy.</p>

CC BY-SA 4.0
2021-09-12T13:19:24.537Z
I would agree for recent autopolyploids, but probably will be less true for polyploids with divergent haplotypes (e.g. allopolyploids).
Kamil S Jaron
CC BY-SA 4.0
2021-09-12T20:37:52.613Z
@KamilSJaron There is not a good way to assemble those genomes for the moment and therefore few know what coverage is needed anyway. 30X would still be a good starting point.
user172818
CC BY-SA 4.0
2021-09-11T02:09:57.937Z
2021-09-11T02:09:57.937Z
user172818
<p>I am reading the <a href="https://www.pacb.com/wp-content/uploads/Application-Brief-Whole-genome-sequencing-for-de-novo-assembly-Best-Practices.pdf" rel="nofollow noreferrer">Whole Genome Sequencing for <em>de novo</em> Assembly Best Practices</a></p>
<blockquote>
<ul>
<li>Use the Sequel II or IIe System and SMRT® Cell 8M to sequence to desired coverage depth for complexity
of genome</li>
<li>10- to 15-fold coverage per haplotype recommended</li>
</ul>
</blockquote>
<p>First of all Wikipedia shows different definitions for the term <a href="https://en.wikipedia.org/wiki/Haplotype" rel="nofollow noreferrer">haplotype</a>. Which one applies here?</p>
<p>Second while the recommendations state to use <code>10- to 15-fold</code> coverage per haplotype I find myself confused about how I would know the <a href="https://en.wikipedia.org/wiki/Haplotype" rel="nofollow noreferrer">haplotype</a> beforehand.</p>
<p>From what I understand of <a href="https://en.wikipedia.org/wiki/Haplotype_estimation" rel="nofollow noreferrer">haplotype-estimation</a> I would need the sequenced data first.</p>
<p>How would I go about this when wanting to do a <em>de novo</em> assembly of a species that has no published assembly of any close relatives? That means the only information I would have at that point is the C-value of the species to be sequenced.</p>
<p>Or better, how does the Sequel II figure out the number of haplotypes for achieving the desired fold-coverage?</p>
<p>I hope I'm in the right SE.</p>

<assembly><coverage><pacbio>
How do you set the coverage in PacBio's Sequel II?
# How do you set the coverage in PacBio's Sequel II?

(Asked by: username_0 on 2021-09-09)

I am reading the [Whole Genome Sequencing for *de novo* Assembly Best Practices](https://www.pacb.com/wp-content/uploads/Application-Brief-Whole-genome-sequencing-for-de-novo-assembly-Best-Practices.pdf)

> * Use the Sequel II or IIe System and SMRT® Cell 8M to sequence to desired coverage depth for complexity
>   of genome
> * 10- to 15-fold coverage per haplotype recommended

First of all Wikipedia shows different definitions for the term [haplotype](https://en.wikipedia.org/wiki/Haplotype). Which one applies here?

Second while the recommendations state to use `10- to 15-fold` coverage per haplotype I find myself confused about how I would know the [haplotype](https://en.wikipedia.org/wiki/Haplotype) beforehand.

From what I understand of [haplotype-estimation](https://en.wikipedia.org/wiki/Haplotype_estimation) I would need the sequenced data first.

How would I go about this when wanting to do a *de novo* assembly of a species that has no published assembly of any close relatives? That means the only information I would have at that point is the C-value of the species to be sequenced.

Or better, how does the Sequel II figure out the number of haplotypes for achieving the desired fold-coverage?

I hope I'm in the right SE.

**Tags:** assembly, coverage, pacbio

---

## 2 answers

### Answer by username_1 on 2021-09-09. Score: 2 | Accepted answer

They just want to know the ploidy of the sample. In a diploid sample, e.g. humans, there are two haplotypes. This is mostly important for haplotype resolution (i.e. get the two full genomes in a diploid).

Note that elsewhere in the brochure that you link it writes:

> Fast and efficient assembly of even
> the largest genomes with haplotype
> resolution of complex polyploids

If you are working with a diploid species, that translates to 20-30X coverage. That seems low to me for the older tech of CLR/Sequel sequencing, but they are the experts.

Different recommendations posted [here](https://pacbio.gs.washington.edu/) by a service provider suggest 40-50X for structural variant analysis, which seems more reasonable.

> **Comments:**
> **username_0**: There are still situations where I might not know the ploidity due to a lack of data. At least for now I was not able to find any data on either phylum: Cnidaria or  the class: Scyphozoa or the order: Rhizostomeae. Or in the case of *Sequoia sempervirens* how did they estimate the ploidity in advance?  Thanks for the insights!  
> **username_1**: @ilamengl I think that there are some methods you can look into: 1) karyotype analysis, 2) computational tools, here is one example: https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-018-2128-z  
> **username_1**: Additionally, there is a ploidy database which does appear to have cnidarians: https://cromanpa94.github.io/ACC/  

---

### Answer by username_2 on 2021-09-11. Score: 1

Unless you want to reconstruct all haplotypes, you can sequence to ~30X regardless of ploidy.

> **Comments:**
> **username_3**: I would agree for recent autopolyploids, but probably will be less true for polyploids with divergent haplotypes (e.g. allopolyploids).  
> **username_2**: @KamilSJaron There is not a good way to assemble those genomes for the moment and therefore few know what coverage is needed anyway. 30X would still be a good starting point.  

---

