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What coverage is recommended for amplicon sequencing?
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12 months ago
Picasa • 390

Hello,

My goal is to make a gene tree of different genes and multiple species. I will amplify genes via pcr and get amplicon sequencing.

Do you have any recommendation for the sequencing coverage ?

Thanks.

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I would recommend 100% coverage of your target region.

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I assume you are not thinking high-throughput sequencing here. If you just need a representative sequence from each gene and species?

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Yes I need a representative sequence for each gene and species but my idea was more like sequencing each gene for each species at a certain coverage (how much, is my question) with high-throughput sequencing and get the "most represented".

Example for one gene of one species, if I have a coverage of 5X (it's a random number, I am asking what is usually do):

ATGCGT
ATGCGT
ATGCGT
ATGCGT
ATGCGA

I would choose the gene with this sequence : ATGCGT because it is represented 4 times among 5.

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Okay you're asking about depth of coverage (I assumed breadth of coverage, see my comment, my bad!). Depth of coverage depends on many factors, such as which platform you use, and how many genes you have (and how many reads are necessary for 1 X depth of coverage of your whole targets region), and how many reads are available (per lane or per chip in one run). It also depends on if you only expect homozygous and heterozygous alleles in your samples, or also somatic mutations. For the first two, I would recommend at least 50 or 100 X per allele at minimum.

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Oh sorry, I will detail more. 50x or 100x seems quite a lot no ?

I have 7 genes and 200 individual of the same species (but different populations), so a total of 1400 genes to anaylse.

The genes are quite short (500 bp in average).

And my goal is to make a tree of genes.

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