Biodiversity Data Journal :
Research Article
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Corresponding author: Seikan Kurata (kangrookangaeru@gmail.com)
Academic editor: Xin Zhou
Received: 07 Dec 2023 | Accepted: 25 May 2024 | Published: 18 Jun 2024
© 2024 Seikan Kurata, Shota Mano, Naoyuki Nakahama, Shun Hirota, Yoshihisa Suyama, Motomi Ito
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kurata S, Mano S, Nakahama N, Hirota S, Suyama Y, Ito M (2024) Development of mitochondrial DNA cytochrome c oxidase subunit I primer sets to construct DNA barcoding library using next-generation sequencing. Biodiversity Data Journal 12: e117014. https://doi.org/10.3897/BDJ.12.e117014
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Insects are one of the most diverse eukaryotic groups on the planet, with one million or more species present, including those yet undescribed. The DNA barcoding system has been developed, which has aided in the identification of cryptic species and undescribed species. The mitochondrial cytochrome c oxidase I region (mtDNA COI) has been utilised for the barcoding analysis of insect taxa. Thereafter, next-generation sequencing (NGS) technology has been developed, allowing for rapid acquisition of massive amounts of sequence data for genetic analyses. Although NGS-based PCR primers designed to amplify the mtDNA COI region have been developed, their target regions were only a part of COI region and/or there were taxonomic bias for PCR amplification. As the mtDNA COI region is a traditional DNA marker for the DNA barcoding system, modified primers for this region would greatly contribute to taxonomic studies. In this study, we redesigned previously developed PCR primer sets that targetted the mtDNA COI barcoding region to improve amplification efficiency and to enable us to conduct sequencing analysis on NGS. As a result, the redesigned primer sets achieved a high success rate (> 85%) for species examined in this study, covering four insect orders (Coleoptera, Lepidoptera, Orthoptera and Odonata). Thus, by combining the primers with developed primer sets for 12S or 16S rRNA regions, we can conduct more detailed taxonomic, phylogeographic and conservation genetic studies using NGS.
biodiversity, DNA barcoding, mtDNA COI, insect, next-generation sequencing
Biodiversity can be categorised into three levels: species diversity (richness), genetic diversity and ecosystem diversity. Species richness provides a straightforward method for describing community and regional diversity (
The DNA barcoding system was developed to identify species through DNA sequencing (
Although primer sets have been developed to amplify the COI region for NGS-based analysis (i.e. DNA metabarcoding; for example,
To identify polymorphic sites in the primer annealing regions of different insect taxa, we downloaded COI sequences from the NCBI database (Suppl. material
Primer name |
Primer sequence (5’ –3’)* |
Target region |
LCO1490 |
TCWACWAAYCAYAARGAYATYGG |
1–319 bp of the mtDNA COI |
COmfd_R |
GGDGGRTANAHHGTTCAHCCNGTHCC |
|
COmfd_F |
CCNCGRHTRAAYAAYATRAGWTTYTG |
262–658 bp of the mtDNA COI |
HCO2198 |
ACTTCDGGRTGNCCAAARAAYCA |
Between April to October 2022, we collected 96 specimens comprising 96 species, 48 families and 11 orders (Table
Specimen samples information |
Number of reads* |
Sampling location |
|||||
Order |
Family |
species |
1-319 bp |
262-658 bp |
Locality name |
Latitude |
Longitude |
Coleoptera |
Carabidae |
Carabus vanvolxemi Putzeys, 1875 |
2839 |
3520 |
Japan: Mt. Moriyoshi |
|
|
Carabidae |
Carabus insulicola Chaudoir, 1869 |
4035 |
2954 |
Japan: Akita, Yamamoto, Mitane |
|
|
|
Carabidae |
Craspedonotus tibialis Schaum, 1863 |
149 |
102 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Carabidae |
Cylindera ovipennis Bates, 1883 |
131 |
215 |
Japan: Akita, Akita, Nibetsu |
|
|
|
Carabidae |
Scarites terricola Bonelli, 1813 |
6141 |
2384 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Carabidae |
Pterostichus Bonelli, 1810 |
3746 |
1547 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Ceranbycidae |
Acalolepta luxuriosa Bates, 1873 |
130 |
24 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Ceranbycidae |
Anoplophora chinensis Forster, 1771 |
18024 |
15712 |
Japan: Akita, Akita, Nibetsu |
|
|
|
Ceranbycidae |
Batocera lineolata Chevrolat, 1852 |
7707 |
2946 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Ceranbycidae |
Prionus insularis Motschulsky, 1857 |
980 |
610 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Coccinellidae |
Aiolocaria hexaspilota Hope, 1831 |
23 |
24 |
Japan: Aktia, Akita, Toyoiwatoyomaki |
|
|
|
Dytiscidae |
Cybister chinensis Motschulsky, 1854 |
109 |
2 |
Japan: Akita, Noshiro, Asanai |
|
|
|
Dytiscidae |
Graphoderus adamsii Clark, 1864 |
57 |
9 |
Japan: Akita, Noshiro, Asanai |
|
|
|
Dytiscidae |
Rhantus suturalis MacLeay, 1825 |
- |
- |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Hydrophilidae |
Hydrochara affinis Sharp, 1873 |
8020 |
7971 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Hydrophilidae |
Hydrophilus acuminatus Motschulsky, 1854 |
8335 |
11208 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Lucanidae |
Dorcus rectus Motschulsky, 1858 |
326 |
835 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Lucanidae |
Prosopocoilus inclinatus Motschulsky, 1857 |
1184 |
2856 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Rhynchophoridae |
Sipalinus gigas Fabricius, 1775 |
2783 |
1162 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Scarabaeidae |
Anomala schoenfeldti Ohaus, 1915 |
536 |
337 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Scarabaeidae |
Polyphylla albolineata Motschulsky, 1861 |
1525 |
3223 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Scarabaeidae |
Protaetia orientalis Gory & Percheron, 1833 |
5218 |
7267 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Scarabaeidae |
Trypoxylus dichotomus Linnaeus, 1771 |
1673 |
1417 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Tenebrionidae |
Cryphaeus amurensis Heyden, 1884 |
- |
- |
Japan: Akita, Yamamoto, Mitane |
|
|
|
Hymenoptera |
Apidae |
Apis mellifera Linnaeus, 1758 |
66 |
1727 |
Japan: Akita, Akita, Shimoshinjo |
|
|
Apidae |
Bombus diversus Smith, 1869 |
608 |
727 |
Japan: Aomori, Shimokita, Higashidori |
|
|
|
Apidae |
Bombus terrestris Linnaeus, 1758 |
- |
- |
Japan: Hokkaido, Hakodate, Bandai |
|
|
|
Pompilidae |
Auplopus carbonarius Scopoli, 1763 |
- |
- |
Japan: Mt. Moriyoshi |
|
|
|
Scoliidae |
Scolia histrionica Smith, 1873 |
- |
- |
Japan: Aomori, Shimokita, Higashidori |
|
|
|
Vespidae |
Oreumenes decoratus Smith, 1852 |
443 |
268 |
Japan: Yamagata, Tsuruoka, Obari |
|
|
|
Vespidae |
Parapolybia indica Saussure, 1854 |
1612 |
1106 |
Japan: Akita, Akita, Kanaashi |
|
|
|
Vespidae |
Polistes chinensis antennalis Yamane, 1972 |
26 |
9 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Vespidae |
Polistes jokahamae Radoszkowski, 1887 |
3867 |
3411 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Vespidae |
Polistes rothneyi Cameron, 1900 |
626 |
338 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Vespidae |
Vespa analis Fabricius, 1775 |
1090 |
868 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Vespidae |
Vespa ducalis Smith, 1852 |
1572 |
1669 |
Japan: Yamagata, Shinjo, Tsunozawa |
|
|
|
Vespidae |
Vespa mandarinia Smith, 1852 |
7871 |
12385 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Lepidoptera |
Agaristidae |
Sarbanissa subflava Moore, 1877 |
4388 |
2931 |
Japan: Akita, Minamiakita, Ogata |
|
|
Callidulidae |
Pterodecta felderi Bremer, 1864 |
3011 |
8868 |
Japan: Yamagata, Tsuruoka, Obari |
|
|
|
Drepanidae |
Hypsomadius insignis Butler, 1877 |
858 |
1338 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Heterogeneidae |
Parasa sinica Moore, 1877 |
3105 |
1538 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Lycaenidae |
Lycaena phlaeas Linnaeus, 1761 |
48 |
- |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Lycaenidae |
Hypaurotis fujisanus Matsumura, 1910 |
1124 |
307 |
Japan: Yamagata, Tsuruoka, Obari |
|
|
|
Lymantriidae |
Euproctis similis Fuessly, 1775 |
36 |
19 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Notodontidae |
Pterostoma gigantina Staudinger, 1892 |
1110 |
65 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Nymphalidae |
Nymphalis canace Linnaeus, 1763 |
2009 |
1002 |
Japan: Aichi, Tahara, Nishiyama |
|
|
|
Nymphalidae |
Minois dryas Scopoli, 1763 |
1000 |
503 |
Japan: Aomori, Kitatsugaru, Nakadomari |
|
|
|
Nymphalidae |
Neope niphonica Butler, 1881 |
10303 |
8532 |
Japan: Mt. Moriyoshi |
|
|
|
Nymphalidae |
Ochlodes ochracea Bremer, 1861 |
1295 |
2339 |
Japan: Iwate, Kunohe, Kunohe |
|
|
|
Nymphalidae |
Vanessa indica Herbst, 1794 |
12816 |
5480 |
Japan: Yamagata, Tsuruoka, Wasada |
|
|
|
Papilionidae |
Papilio xuthus Linnaeus, 1767 |
1019 |
1520 |
Japan: Aichi, Tahara, Nishiyama |
|
|
|
Papilionidae |
Parnassius citrinarius Butler, 1866 |
8379 |
11203 |
Japan: Yamagata, Tsuruoka, Wasada |
|
|
|
Pieridae |
Colias erate Esper, 1805 |
6181 |
1115 |
Japan: Akita, Yurihonjo, Tsuchiya |
|
|
|
Pieridae |
Pieris melete Menetries, 1857 |
4541 |
6119 |
Japan: Iwate, Kunohe, Kunohe |
|
|
|
Saturniidae |
Saturnia japonica Moore, 1872 |
4500 |
8871 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Sphindidae |
Agrius convolvuli Linnaeus, 1758 |
5921 |
10323 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Sphindidae |
Smerinthus planus Walker, 1856 |
4596 |
703 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Hemiptera |
Acanthosomatidae |
Sastragala esakii Hasegawa, 1959 |
137 |
33 |
Japan: Akita, Akita, Nibetsu |
|
|
Belostomatidae |
Appasus japonicus Vuillefroy, 1864 |
201 |
641 |
Japan: Aktia, Akita, Toyoiwatoyomaki |
|
|
|
Belostomatidae |
Appasus major Esaki, 1934 |
88 |
93 |
Japan: Aktia, Akita, Toyoiwatoyomaki |
|
|
|
Cicadellidae |
Bothrogonia ferruginea Fabricius, 1787 |
150 |
26 |
Japan: Yamagata, Tsuruoka, Wasada |
|
|
|
Cicadidae |
Graptopsaltria nigrofuscata Motschulsky, 1866 |
6556 |
7179 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Cicadidae |
Platypleura kaempferi Fabricius, 1794 |
3304 |
3833 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Cicadidae |
Yezoterpnosia nigricosta Motschulsky, 1866 |
17050 |
18002 |
Japan: Mt. Moriyoshi |
|
|
|
Coreidae |
Leptoglossus occidentalis Heidemann, 1910 |
404 |
74 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Corixidae |
Hesperocorixa hokkensis Matsumura, 1905 |
- |
- |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Gerridae |
Aquarius paludum Fabricius, 1794 |
- |
- |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Notonectidae |
Notonecta triguttata Motschulsky, 1861 |
621 |
416 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Pentatomidae |
Palomena angulosa Motschulsky, 1861 |
- |
- |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Pentatomidae |
Pentatoma japonica Distant, 1882 |
- |
8 |
Japan: Iwate, Kunohe, Kunohe |
|
|
|
Reduviidae |
Agriosphodrus dohrni Stal, 1862 |
- |
- |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Reduviidae |
Ectrychotes andreae Thunberg, 1784 |
65 |
- |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Reduviidae |
Velinus nodipes Uhler, 1860 |
- |
- |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Orthoptera |
Acrididae |
Acrida cinerea Thunberg, 1815 |
5446 |
9947 |
Japan: Akita, Akita, Shimoshinjo |
|
|
Acrididae |
Aiolopus thalassinus Fabricius, 1781 |
13476 |
7387 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Acrididae |
Locusta migratoria Linnaeus, 1758 |
2739 |
452 |
Japan: Aichi, Tahara, Nishiyama |
|
|
|
Acrididae |
Oedaleus infernalis Saussure, 1884 |
1095 |
441 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Gryllotalpidae |
Gryllotalpa orientalis Burmeister, 1838 |
2842 |
3479 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Tettigoniidae |
Ruspolia dubia Redtenbacher, 1891 |
271 |
317 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Diptera |
Asilidae |
Neoitamus angusticornis Loew, 1858 |
- |
- |
Japan: Akita, Akita, Shimoshinjo |
|
|
Asilidae |
Neoitamus Osten Sacken, 1878 |
16 |
5 |
Japan: Mt. Moriyoshi |
|
|
|
Asilidae |
Promachus yesonicus Bigot, 1887 |
3003 |
2058 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Tabanidae |
Tabanus chrysurus Loew, 1858 |
144 |
79 |
Japan: Iwate, Morioka, Yabukawa |
|
|
|
Odonata |
Coenagrionidae |
Paracercion hieroglyphicum Brauer, 1865 |
871 |
928 |
Japan: Akita, Katagami, Ten-nou |
|
|
Gomphidae |
Davidius nanus Selys, 1869 |
1009 |
1060 |
Japan: Iwate, Kunohe, Kunohe |
|
|
|
Lestidae |
Lestes sponsa Hansemann, 1823 |
31 |
3 |
Japan: Iwate, Morioka, Yabukawa |
|
|
|
Libellulidae |
Rhyothemis fuliginosa Selys, 1883 |
2078 |
2880 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Libellulidae |
Sympetrum darwinianum Selys, 1883 |
87 |
107 |
Japan: Akita, Minamiakita, Ogata |
|
|
|
Libellulidae |
Sympetrum frequens Selys, 1883 |
999 |
1698 |
Japan: Akita, Akita, Sotoasahikawa |
|
|
|
Libellulidae |
Sympetrum infuscatum Selys, 1883 |
268 |
712 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Libellulidae |
Sympetrum kunckeli Selys, 1884 |
449 |
1340 |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Dermaptera |
Labiduridae |
Labidura riparia Pallas, 1773 |
- |
39 |
Japan: Akita, Akita, Shimoshinjo |
|
|
Neuroptera |
Myrmeleontidae |
Myrmeleon bore Tjeder, 1941 |
160 |
41 |
Japan: Akita, Minamiakita, Ogata |
|
|
Mantodea |
Mantidae |
Statilia maculata Thunberg, 1784 |
1328 |
156 |
Japan: Akita, Minamiakita, Ogata |
|
|
Mantidae |
Tenodera aridifolia Stoll, 1813 |
46 |
- |
Japan: Akita, Akita, Shimoshinjo |
|
|
|
Blattaria |
Blattidae |
Periplaneta japonica Karny, 1908 |
395 |
631 |
Japan: Akita, Akita, Shimoshinjo |
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Genomic DNA was extracted using DNeasy Blood & Tissue Kits (QIAGEN, Hilden, Germany) and the total DNA concentration was quantified with a NanoDrop ND-1000 (Thermo Scientific, Waltham, USA). Polymerase chain reaction (PCR) was performed for each specimen according to the manufacturer’s protocol, in a final volume of 10 µl that included 5–10 ng of DNA, 1.0 µl Ex Taq Buffer, 0.2 µmol/l of primers, 0.8 µl of dNTP mixture (2.5 mM of each dNTP), 2 U of Takara Ex Taq polymerase (Takara Bio, Otsu, Japan) and sterile distilled water up to 10 µl. The PCR thermal cycling conditions were an initial 1 min denaturation at 94°C; 35 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 1 min, with a final 20-min extension at 72°C. The PCR product was verified using a MultiNA microchip electrophoresis system (SHIMADZU, Kyoto, Japan). Before sequencing, the PCR products (i.e. LCO1490–COmfd_R and COmfd_F–HCO2198) were pooled for each specimen sample and all 96 samples were prepared for sequencing. Subsequent paired-end sequencing was conducted using 2 × 250 bp cycle run on an Illumina MiSeq Sequencer (Illumina, San Diego, USA) and with the MiSeq Reagent Nano Kit v.2 (500 cycles).
Data preprocessing, quality control and identification of representative sequences were conducted using Claident v.0.2.2019.05.10 (
Before quality control and data analysis using Claident, non-demultiplexed fastq files (261 bp) were produced from BCL files using bcl2fastq v.1.8.4 (Illumina). During this step, non-demultiplexed fastq reads were sorted, based on index reads (index1: 9 bp, index2: 5 bp) and the last position of the raw reads was trimmed (--use-bases-mask Y260n,I9,I5,Y260n). Subsequently, the non-multiplexed fastq reads were demultiplexed using the Claident command clsplitseq, which specifies indices and primer sequences and the quality threshold of the index sequence was set to 30 (--minqualtag=30). As the modified primers included 0–3 Ns for accommodation, the option (--truncateN=enable) was used. Files output from clsplitseq were deposited in the DDBJ (accession no. DRA017438).
As paired-end sequencing was used, it was possible to identify overlaps between forward and reverse reads. The clconcatpair command with the --mode=OVL argument was used to generate concatenated reads from the forward and reverse sequences. Any low-quality reads were filtered out using the clfilterseq command with settings --maxplowequal=0.1 --minqual=27, to remove positions with quality lower than Q27.
Further cleaning to remove noisy and chimeric sequences was performed using the following clcleanseqv parameters: --derepmode=FULLLENGTH --primarymaxnmismatch=0 --secondarymaxnmismatch=1 --pnoisycluster=0.5. Representative sequences for each sample were identified using the clclasseqv command with a 99% identity threshold (--minident=0.99). However, the sequences output by clclasseqv remained separated into two regions.
Overlap was detected and the sequences from the two loci were merged using the EMBOSS programme (
We redesigned two primer sets (LCO1490–COmfd_R and COmfd_F–HCO2198) that amplified the DNA barcoding region of the mtDNA COI region of insect taxa (see Table
Using the modified COI primer sets for DNA barcoding, we conducted PCR to amplify samples from 96 species, encompassing 48 families and 11 orders and performed NGS sequencing analysis. These primers successfully amplified and sequenced the target mtDNA COI regions of 80 species from 41 families in 11 orders. Notably, the primer sets had high success rates for Coleoptera, Lepidoptera, Orthoptera and Odonata (Table
Order |
No. of specimen samples* |
No. of samples* |
Success rate (%) |
Coleoptera |
24 |
22 |
91.7 |
Hymenoptera |
14 |
10 |
71.4 |
Lepidoptera |
20 |
19 |
95 |
Hemiptera |
16 |
9 |
56.3 |
Orthoptera |
6 |
6 |
100 |
Diptera |
4 |
3 |
75 |
Odonata |
9 |
8 |
88.9 |
Others |
5 |
3 |
– |
Total |
96 |
80 |
82.3 |
After the clustering step in Claident, the modified barcoding primers generated 12–18024 (median, 1102; average, 2774) and 2–18002 (median, 1060; average, 2720) reads using LCO1490–COmfd_R and COmfd_F–HCO2198, respectively (Table
To assess whether the modified barcoding primer sets could differentiate various insect taxa, we conducted phylogenetic analysis. The resulting phylogenetic tree showed that related insect taxa clustered within the same lineages (Fig.
The Maximum-Likelihood (ML) phylogenetic tree, based of mtDNA COI region which originated from sequence reads of the modified primer sets. The numbers on the major branches represents bootstrap values from the ultrafast bootstrap replications and SH-aLRT methods, respectively. The horizontal scale bar under the tree represents evolutionary distance between specimen taxa. Headers of scientific names represent abbreviations of the order names (e.g. Col. and Hym.).
The goal of our research was to modify the existing mtDNA COI primer set established by
Advances in NGS system have led to various NGS applications in ecological studies.
The mtDNA COI region is considered challenging for designing new NGS-based primer sets due to the high polymorphism rate (
The authors thank Drs. Junsuke Yamasako and Natsuko Kondo for granting access to their collection materials. We also thank the reviewer for their insightful comments and suggestions to revise our manuscript.
The National BioResource Project
18km0210136j0002
Seikan Kurata, Naoyuki Nakahama and Motomi Ito conceived the ideas. Shun K. Hirota and Yoshihisa Suyama performed sequencing analysis. Seikan Kurata, Shota Mano and Shun K. Hirota contributed to interpret the analysis results and data curration. All authors contributed to write the manuscript.
The authors declare that they have no conflicts of interest.
We designed the PCR primers by aligning the sequences.
The neighbour-joining (NJ) tree based of mtDNA COI region which originated from sequence reads of the modified primer sets.
The number of reads from first half (LCO1490–COmfd_R: 1-319) and second half (COmfd_F–HCO2198: 262-658) of the COI region. There is a great difference for the number of reads between the most dominant and the second largest read. In addition, the results of the BLAST search were shown on the right side in the Table.
Comparison results of Chironomid NGS assemblies against the sequence data from Sanger sequencing.
Illumina adapter sequences (F: CGCTCTTCCGATCTCTG; R: TGCTCTTCCGATCTGAC) were added to the 5’ end of the primer sequence and 0–3Ns were inserted between adapter sequences and primer sequences.
The number of reads from first half (LCO1490–COmfd_R: 1-319) and second half (COmfd_F–HCO2198: 262-658) of the COI region, which were clustered and counted by Claident and the unit is base pair (bp). The symbol of “–” indicates that suitable fragment reads for the mtDNA COI region were not detected.
The number of specimen samples for sequencing analysis.
The number of samples which pass the PCR trials and NGS-based sequencing analysis.