DNA metabarcoding of insects and allies: an evaluation of primers and pipelines.
Metabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80-90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.
Museum of Zoology,Institute of Biological Sciences,Faculty of Science,University of Malaya,50603 Kuala Lumpur,Malaysia.
G-J
GJ
Brandon-Mong
School of Science,Monash University Malaysia,Jalan Lagoon Selatan,Bandar Sunway,47500 Petaling Jaya,Selangor,Malaysia.
H-M
HM
Gan
Museum of Zoology,Institute of Biological Sciences,Faculty of Science,University of Malaya,50603 Kuala Lumpur,Malaysia.
K-W
KW
Sing
Museum of Zoology,Institute of Biological Sciences,Faculty of Science,University of Malaya,50603 Kuala Lumpur,Malaysia.
P-S
PS
Lee
Institute of Ocean and Earth Sciences (IOES),University of Malaya,50603 Kuala Lumpur,Malaysia.
P-E
PE
Lim
Museum of Zoology,Institute of Biological Sciences,Faculty of Science,University of Malaya,50603 Kuala Lumpur,Malaysia.
J-J
JJ
Wilson
D000818:Animals; D001181:Arthropods / Q000235:genetics*; D044822:Biodiversity; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D056186:Metagenomics; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004272:DNA, Mitochondrial; D003576:Electron Transport Complex IV
Arthropoda; COI; Illumina MiSeq; Malaise trap; biodiversity; high-throughput sequencing