Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species
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INTRODUCTION: Invertebrate communities are central to many environmental monitoring programs. In freshwater ecosystems, aquatic macroinvertebrates are collected, identified and then used to infer ecosystem condition. Yet the key step of species identification is often not taken, as it requires a high level of taxonomic expertise, which is lacking in most organizations, or species cannot be identified as they are morphologically cryptic or represent little known groups. Identifying species using DNA sequences can overcome many of these issues; with the power of next generation sequencing (NGS), using DNA sequences for routine monitoring becomes feasible. RESULTS: In this study, we test if NGS can be used to identify species from field-collected samples in an important bioindicator group, the Chironomidae. We show that Cytochrome oxidase I (COI) and Cytochrome B (CytB) sequences provide accurate DNA barcodes for chironomid species. We then develop a NGS analysis pipeline to identifying species using megablast searches of high quality sequences generated using 454 pyrosequencing against comprehensive reference libraries of Sanger-sequenced voucher specimens. We find that 454 generated COI sequences successfully identified up to 96% of species in samples, but this increased up to 99% when combined with CytB sequences. Accurate identification depends on having at least five sequences for a species; below this level species not expected in samples were detected. Incorrect incorporation of some multiplex identifiers (MID's) used to tag samples was a likely cause, and most errors could be detected when using MID tags on forward and reverse primers. We also found a strong quantitative relationship between the number of 454 sequences and individuals showing that it may be possible to estimate the abundance of species from 454 pyrosequencing data. CONCLUSIONS: Next generation sequencing using two genes was successful for identifying chironomid species. However, when detecting species from 454 pyrosequencing data sets it was critical to include known individuals for quality control and to establish thresholds for detecting species. The NGS approach developed here can lead to routine species-level diagnostic monitoring of aquatic ecosystems.
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Small organisms may be lost owing to low number of sequence reads.
“small organisms may be lost owing to low number of sequence reads [38].”
Sampled organisms have vastly different biomasses.
“First, sampled organisms have vastly different biomasses, and thus small organisms may be lost owing to low number of sequence reads [38].”
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- Authors
- Carew, M. E., Pettigrove, V. J., Metzeling, L., & Hoffmann, A. A.
- Affiliations
- The University of Melbourne (AU)
- Environment Protection Authority Victoria (AU)
- Corresponding author
- Carew, Melissa E
- Venue
- Frontiers in Zoology
- Published
- 2013
- Volume
- 10
- Issue
- 1
- Pages
- 45
- ISSN
- 1742-9994 (ISSN-L)
- PMC
- PMC3750358
- OpenAlex
- W2159396143
- Cited by
- 243
- References
- 76
- Citations by year
- 2026: 5 · 2025: 4 · 2024: 5 · 2023: 12 · 2022: 8 · 2021: 16 · 2020: 27 · 2019: 23 · 2018: 50 · 2017: 33 · 2016: 24 · 2015: 24 · 2014: 11
- FWCI
- 17.38
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- Frontiers in Zoology (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Minerva Access (University of Melbourne) (submittedVersion, cc-by)
- Funders
- Melbourne Water
- State Government of Victoria
- Environment Protection Authority Victoria
- Australian Research Council
- Topics
- Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
- Life Sciences › Agricultural and Biological Sciences › Ecology, Evolution, Behavior and Systematics › Invertebrate Taxonomy and Ecology
- Physical Sciences › Environmental Science › Ecology › Freshwater macroinvertebrate diversity and ecology
- Keywords
- DNA barcoding, Chironomidae, aquatic macroinvertebrates, bioindicator species, species identification, COI, cytochrome b, 454 pyrosequencing, environmental monitoring, freshwater ecosystems, species abundance estimation, reference library
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