Strengths and limits of long read metabarcoding
Résumé
Metabarcoding is the large-scale taxonomic identification of complex environmental samples via analysis of DNA reads of one marker gene. Different marker genes are used, the 16S rRNA gene is mainly used to identify bacteria. We conducted a comparative study to understand the limitations and strengths of PacBio HiFi long read sequencing technology for metabarcoding analyses. We performed the same analyses on two types of datasets. A commercial ZymoBIOMICS mock community consisting of eight bacteria and 32 samples containing pig fecal microbiota from the ExpomycoPig project. We focused on the 16S-23S rRNA gene operon and sequenced 3 types of amplicons by metabarcoding: the V3-V4 region of the 16S rRNA, the full-length 16S rRNA gene and the full-length 16S-23S gene operon. We observe that by using HIFI long reads we increase the specificity of sample characterization. But when we analyze the 16S-23S amplicons, the specificity increases within the limit of the availability of the sequence in the databases that are much less comprehensive than the databases containing only 16S data. A strategy of affiliating 16S-23S amplicons on complementary databases (Silva 16S and 23S and a custom 16S-23S operon database) improves microbial identifications.
Domaines
Bio-informatique [q-bio.QM]Origine | Fichiers produits par l'(les) auteur(s) |
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