10.5061/DRYAD.G05G074
Akankunda, Trace
University of Adelaide
To, Hien
Norwegian University of Life Sciences
Lopez, Carlos R
University of Kentucky
Leijs, Remko
University of Adelaide
Hogendoorn, Katja
0000-0002-4942-8062
University of Adelaide
Data from: A method to generate multi-locus barcodes of pinned insect
specimens using MiSeq
Dryad
dataset
2019
Insect collections
Multilocus barcoding
The Holsworth Wildlife Research Endowment Fund and the Australian
Government Department of Agriculture and Water Resources
15-02-035
2020-01-15T00:00:00Z
2020-01-15T00:00:00Z
en
https://www.ncbi.nlm.nih.gov/sra/SRP151814.
2980705 bytes
3
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
For molecular insect identification, amplicon sequencing methods are
recommended because they offer a cost effective approach for targeting
small sets of informative genes from multiple samples. In this context,
high-throughput multilocus amplicon sequencing has been achieved using the
MiSeq Illumina sequencing platform. However, this approach generates short
gene fragments of less than 500 bp, which then have to be overlapped using
bioinformatics to achieve longer sequence lengths. This increases the risk
of generating chimeric sequences or leads to the formation of incomplete
loci. Here, we propose a modified nested amplicon sequencing method for
targeting multiple loci from pinned insect specimens using the MiSeq
Illumina platform. The modification exists in using a three-step nested
PCR approach targeting near full length loci in the initial PCR and
subsequently amplifying short fragments between 300 and 350 bp for
high-throughput sequencing using Illumina chemistry. Using this method, we
generated 407 barcode-compliant sequences for three loci from 98 of 138
pinned specimens. This method worked best for pinned specimens aged
between 0 - 5 years, with a limit of 10 years for pinned and 14 years for
ethanol preserved specimens. Hence, our method overcomes some of the
challenges of amplicon sequencing using short read Next Generation
Sequencing and improves possibilities to create high quality multilocus
barcodes from insect collections.
GenBank blast search results for the consensus sequences generated in this
study Folder contains GenBank blast search results for consensus sequences
of 3 genes Cytochrome Oxidase 1 (5' and 3'), Elongation Factor 1
alpha (2nd copy) and Wingless gene. The consensus sequences are named in
the format; Species name_sample name_number of reads used to assemble the
consensus sequence. The datasets can be viewed using the Geneious software
program. NestedAmpliconSeqAnalysis-scripts Scripts for inferring consensus
gene sequences of NGS data generated using A novel Nested Amplicon
Sequencing Approach. Also deposited in Github under this link;
https://github.com/UofABioinformaticsHub
DNA was extracted from legs of pinned and ethanol preserved specimens and
amplified using a modified nested PCR approach in order to allow
sequencing on the illumina MiSeq platform. The raw sequence data generated
was processed using custom scripts and the consesus sequences analysed as
described in the manuscript including blast searching against the GenBank
database accessed through the Geneious software program (v 8.1.3).