TY - JOUR

T1 - Short- and long-read metabarcoding of the eukaryotic rRNA operon

T2 - Evaluation of primers and comparison to shotgun metagenomics sequencing

AU - Latz, Meike A.C.

AU - Grujcic, Vesna

AU - Brugel, Sonia

AU - Lycken, Jenny

AU - John, Uwe

AU - Karlson, Bengt

AU - Andersson, Agneta

AU - Andersson, Anders F.

N1 - Publisher Copyright: © 2022 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.

PY - 2022

Y1 - 2022

N2 - High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in applying long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.

AB - High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in applying long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.

KW - marine plankton

KW - metabarcoding

KW - microbial eukaryotes

KW - PacBio long-read sequencing

KW - primer design

KW - rRNA operon

U2 - 10.1111/1755-0998.13623

DO - 10.1111/1755-0998.13623

M3 - Journal article

C2 - 35437888

AN - SCOPUS:85129364555

VL - 22

SP - 2304

EP - 2318

JO - Molecular Ecology

JF - Molecular Ecology

SN - 0962-1083

IS - 6

ER -