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Identify different new strains of a symbiont species (not in the database)

Hi! I'm a PhD student working on insects symbionts. I have a very important query because I really don't know how to proceed with my surveys. I hope you can help me.

Well, I sequenced 16S RNA for different european insects populations and related species (by means of Illumina), and now I have a list of OTUs for Wolbachia (in every population), a very important bacteria involved on insects reproduction. I read many papers about Wolbachia strains, but those were based on Asian insects species, solely. Then, even they found two different Wolbachia strains, its probable that my populations have different strains. Indeed, I aligned the 16S sequences and constructed a neighbour-joining tree, and the result consisted in many clades, without any pattern based on insect populations. Anyway, I ordered the primers that these Asian researchers designed and I'm going to test them on my populations, but in case they don't work I will not know how to proceed and distinguish new strains, I mean, I don't know which level I need to establish in terms of nucleotide substitutions between the 16S Wolbachia sequences and so separate strains.

Thanks in advance!

blast rna-seq alignment next-gen symbionts
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updated by Ram Ram • written by enrique.menriv • enrique.menriv

1 answer

One "established" way of determining bacterial strains is with MLST, which for Wolbachia comprise 5 chosen genes. The reasoning behind MLST is avoiding pitfalls due to genomic recombination, which has been shown to be common in some Wolbachia lineages despite their being intracellular symbionts.

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written by h.mon h.mon

First of all, thank you very much for your answer. I really will opt for these 5 genes, finally. Anyway, I was guessing if I could take advantage of my sequenced 16S ARN samples, that's why I did neighbor-joining tree with them using reference Wolbachia sequences from NCBI, but the output was really bad, and I don't understand why. I put all my sequences in Blast and it gave me 96-99% identity and 100% cover for many Wolbachia sequences. However, in my tree, almost every node has a value of 1 to 4, and my sequences are really far from Wolbachia refseq, and that is really bad. Anyone knows which could be the problem with that?

Thank you again!

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updated by Ram Ram • written by enrique.menriv • enrique.menriv

Did you align the sequences before doing the neighbour-joining?

But you poor tree is probably due to insufficient information to recover the tree with the 16S - did you use only the most similar sequences, or did you pick sequences from several Wolbachia clades and varying degrees of similarity?

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updated by Ram Ram • written by h.mon h.mon

I finally solved the problem. I hadn't read about the need of choosing sequences from different established Wolbachia supergroups, and now I know that I must to figure out in which of them my strains are located by studying genetic divergences. Anyway, as I said, I'm going to do MLST, too. Thank you for your advice!

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updated by Ram Ram • written by enrique.menriv • enrique.menriv

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