A Sequel to Sanger: amplicon sequencing that scales

111

103

DNA metabarcoding is widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints limit most studies to marker lengths below 600 base pairs (bp). Longer sequencing reads of several thousand bp are now possible with third-generation sequencing. Increased marker lengths provide greater taxonomic resolution and allow for phylogenetic methods of classification, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most bioinformatics tools for DNA metabarcoding were designed for short reads and are therefore unsuitable. Here, we used Pacific Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote SSU and LSU rRNA genes and the complete ITS region. We developed an analysis pipeline that reduced error rates to levels comparable to short-read platforms. Validation using a mock community indicated that our pipeline detected 98% of chimeras de novo. We recovered 947 OTUs from water and sediment samples from a natural lake, 848 of which could be classified to phylum, 397 to genus and 330 to species. By allowing for the simultaneous use of three databases (Unite, SILVA and RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 nonfungal OTUs indicate that long-read DNA metabarcoding holds promise for studies of eukaryotic diversity more broadly.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Long‐read DNA metabarcoding of ribosomal RNA in the analysis of fungi from aquatic environments

Heeger

Bourne

Baschien

et al. 2018

111

103

“…DNA barcode studies initially focused on developing the analytical protocols to construct a specimen‐based reference library (Hebert et al, ; Hebert, Penton, Burns, Janzen, & Hallwachs, ). Although improved protocols have reduced costs, leading to the analysis of millions of single specimens (Hajibabaei et al, ; Hebert et al, ; Ivanova, deWaard, & Hebert, ), this approach is too expensive to support large‐scale bio‐monitoring programmes. However, by coupling a DNA barcode reference library with the analytical capacity of high‐throughput sequencers (HTS), DNA metabarcoding provides a path to rapid, low‐cost assessments of species composition (Brandon‐Mong et al, ; Hajibabaei, Shokralla, Zhou, Singer, & Baird, ; Moriniere et al, ; Yu et al, ).…”

Section: Introductionmentioning

confidence: 99%

“…Studies have now employed this approach to assess species composition in communities of aquatic and terrestrial arthropods (Beng et al, 2016;Elbrecht, Vamos, Meissner, Aroviita, & Leese, 2017;Ji et al, 2013), vertebrates (Sato, Sogo, Doi, & Yamanaka, 2017), diatoms (Vasselon et al, 2017) and fungi (Aas, Davey, & Kauserud, 2017;Bellemain et al, 2012;Tedersoo, Tooming-Klunderud, & Anslan, 2018). Such metabarcoding analysis routinely reveals more species than morphological approaches while requiring far less time (Brandon-Mong et al, 2015;Elbrecht, Peinert, & Leese, 2017;Elbrecht, Vamos et al, 2017;Hebert et al, 2018;Ji et al, 2013;Shokralla et al, 2015;Vivien, Lejzerowicz, & Pawlowski, 2016;Yu et al, 2012).…”

mentioning

confidence: 99%

Metabarcoding a diverse arthropod mock community

Braukmann

Ivanova

Prosser

et al. 2019

Self Cite

125

160

Although DNA metabarcoding is an attractive approach for monitoring biodiversity, it is often difficult to detect all the species present in a bulk sample. In particular, sequence recovery for a given species depends on its biomass and mitome copy number as well as the primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 species. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body segments (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR‐amplified separately (Single Leg) and then pooled. The amplicons generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, although the Miseq delivered the highest sequence quality. As expected, species recovery was most efficient from the Single Leg treatment because amplicon abundance varied little among taxa. Among the three treatments where PCR occurred after pooling, the Bulk Abdomen treatment produced a more uniform read abundance than the Bulk Leg or Composite Leg treatment. Primer choice also influenced species recovery and evenness. Our results reveal how variation in protocols can have substantial impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote.

“…As with classic DNA barcoding approaches, incomplete reference databases are often a key factor in causing false negatives. Fortunately, HTS‐based methods have promised feasible paths in producing both standard DNA barcodes (Hebert et al, ; Liu, Yang, Zhou, & Zhou, ; Srivathsan et al, ) and organelle genomes (Straub et al, ; Tang et al, ) at significantly reduced costs. Indeed, large sequencing efforts for plastid genomes have seen significant progress in China.…”

Section: Discussionmentioning

confidence: 99%

Genome‐skimming provides accurate quantification for pollen mixtures

Lang

Tang

et al. 2019

Studies on foraging partitioning in pollinators can provide critical information to the understanding of food‐web niche and pollination functions, thus aiding conservation. Metabarcoding based on PCR amplification and high‐throughput sequencing has seen increasing applications in characterizing pollen loads carried by pollinators. However, amplification bias across taxa could lead to unpredictable artefacts in estimation of pollen compositions. We examined the efficacy of a genome‐skimming method based on direct shotgun sequencing in quantifying mixed pollen, using mock samples (five and 14 mocks of flower and bee pollen, respectively). The results demonstrated a high level of repeatability and accuracy in identifying pollen from mixtures of varied species ratios. All pollen species were detected in all mocks, and pollen frequencies estimated from the number of sequence reads of each species were significantly correlated with pollen count proportions (linear model, R 2 = 86.7%, p = 2.2e−16). For >97% of the mixed taxa, pollen proportion could be quantified by sequencing to the correct order of magnitude, even for species which constituted only 0.2% of the total pollen. In addition, DNA extracted from pollen grains equivalent to those collected from a single honeybee corbicula was sufficient for genome‐skimming. We conclude that genome‐skimming is a feasible approach to identifying and quantifying mixed pollen samples. By providing reliable and sensitive taxon identification and relative abundance, this method is expected to improve our understanding in studies that involve plant–pollinator interactions, such as pollen preference in corbiculate bees, pollen diet analyses and identification of landscape pollen resource use from beehives.