How do you set the coverage in PacBio's Sequel II?

I am reading the Whole Genome Sequencing for de novo Assembly Best Practices

• 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. 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 beforehand.

From what I understand of 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.

2 Answers

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 by a service provider suggest 40-50X for structural variant analysis, which seems more reasonable.

• 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! Sep 9, 2021 at 21:25
• @ilamengl I think that there are some methods you can look into: 1) karyotype analysis, 2) computational tools, here is one example: bmcbioinformatics.biomedcentral.com/articles/10.1186/… Sep 9, 2021 at 22:06
• Additionally, there is a ploidy database which does appear to have cnidarians: cromanpa94.github.io/ACC Sep 9, 2021 at 22:13

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

• I would agree for recent autopolyploids, but probably will be less true for polyploids with divergent haplotypes (e.g. allopolyploids). Sep 12, 2021 at 13:19
• @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. Sep 12, 2021 at 20:37