title: Targeted gene enrichment and high-throughput sequencing for environmental biomonitoring: a case study using freshwater macroinvertebrates.
abstract: Recent studies have advocated biomonitoring using DNA techniques. In this study, two high-throughput sequencing (HTS)-based methods were evaluated: amplicon metabarcoding of the cytochrome C oxidase subunit I (COI) mitochondrial gene and gene enrichment using MYbaits (targeting nine different genes including COI). The gene-enrichment method does not require PCR amplification and thus avoids biases associated with universal primers. Macroinvertebrate samples were collected from 12 New Zealand rivers. Macroinvertebrates were morphologically identified and enumerated, and their biomass determined. DNA was extracted from all macroinvertebrate samples and HTS undertaken using the illumina miseq platform. Macroinvertebrate communities were characterized from sequence data using either six genes (three of the original nine were not used) or just the COI gene in isolation. The gene-enrichment method (all genes) detected the highest number of taxa and obtained the strongest Spearman rank correlations between the number of sequence reads, abundance and biomass in 67% of the samples. Median detection rates across rare (<1% of the total abundance or biomass), moderately abundant (1-5%) and highly abundant (>5%) taxa were highest using the gene-enrichment method (all genes). Our data indicated primer biases occurred during amplicon metabarcoding with greater than 80% of sequence reads originating from one taxon in several samples. The accuracy and sensitivity of both HTS methods would be improved with more comprehensive reference sequence databases. The data from this study illustrate the challenges of using PCR amplification-based methods for biomonitoring and highlight the potential benefits of using approaches, such as gene enrichment, which circumvent the need for an initial PCR step.
authors.affiliation: Cawthron Institute, 98 Halifax Street, 7010, Nelson, New Zealand.|Department of Entomology, Kansas State University, Waters Hall, Manhattan, KS, 66502, USA.
authors.forename: Eddy J
authors.initials: EJ
authors.lastname: Dowle
authors.affiliation: Cawthron Institute, 98 Halifax Street, 7010, Nelson, New Zealand.|Institute of Marine Science, University of Auckland, PO Box 349, Warkworth 0941, New Zealand.
authors.forename: Xavier
authors.initials: X
authors.lastname: Pochon
authors.affiliation: Cawthron Institute, 98 Halifax Street, 7010, Nelson, New Zealand.
authors.forename: Jonathan
authors.initials: J
authors.lastname: C Banks
authors.affiliation: Cawthron Institute, 98 Halifax Street, 7010, Nelson, New Zealand.
authors.forename: Karen
authors.initials: K
authors.lastname: Shearer
authors.affiliation: Cawthron Institute, 98 Halifax Street, 7010, Nelson, New Zealand.|Environmental Research Insitute University of Waikato, Private Bag 3105, 3240 Hamilton, New Zealand.
authors.forename: Susanna A
authors.identifier: http://orcid.org/0000-0003-1976-8266
authors.initials: SA
authors.lastname: Wood
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D058893:DNA Barcoding, Taxonomic / Q000379:methods; D003951:Diagnostic Errors; D004784:Environmental Monitoring / Q000379:methods*; D005618:Fresh Water / Q000469:parasitology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D007448:Invertebrates / Q000145:classification* / Q000235:genetics*; D056186:Metagenomics / Q000379:methods*; D009520:New Zealand; D016133:Polymerase Chain Reaction / Q000379:methods; D045483:Rivers / Q000469:parasitology
chemical_list: D004247:DNA
keywords: biomonitoring; gene enrichment; high-throughput sequencing; macroinvertebrates; metabarcoding