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Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform.

Shokralla S, Porter TM, Gibson JF, Dobosz R, Janzen DH, Hallwachs W, Golding GB, Hajibabaei M - Sci Rep (2015)

Bottom Line: Here, we present a scalable double dual-indexing approach using an Illumina Miseq platform to sequence DNA barcode markers.We achieved 97.3% success by using half of an Illumina Miseq flowcell to obtain 658 base pairs of the cytochrome c oxidase I DNA barcode in 1,010 specimens from eleven orders of arthropods.Our approach recovers a greater proportion of DNA barcode sequences from individuals than does conventional Sanger sequencing, while at the same time reducing both per specimen costs and labor time by nearly 80%.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON, Canada N1G 2W1.

ABSTRACT
Genetic information is a valuable component of biosystematics, especially specimen identification through the use of species-specific DNA barcodes. Although many genomics applications have shifted to High-Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies, sample identification (e.g., via DNA barcoding) is still most often done with Sanger sequencing. Here, we present a scalable double dual-indexing approach using an Illumina Miseq platform to sequence DNA barcode markers. We achieved 97.3% success by using half of an Illumina Miseq flowcell to obtain 658 base pairs of the cytochrome c oxidase I DNA barcode in 1,010 specimens from eleven orders of arthropods. Our approach recovers a greater proportion of DNA barcode sequences from individuals than does conventional Sanger sequencing, while at the same time reducing both per specimen costs and labor time by nearly 80%. In addition, the use of HTS allows the recovery of multiple sequences per specimen, for deeper analysis of genetic variation in target gene regions.

No MeSH data available.


Number and percentage of 1,010 individual arthropod specimens producing a COI DNA sequence that matches morphological identification based on BLAST comparison to public DNA barcode databases.Panel (A) Sanger-generated barcodes. Panel (B) Illumina-generated barcodes.
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f2: Number and percentage of 1,010 individual arthropod specimens producing a COI DNA sequence that matches morphological identification based on BLAST comparison to public DNA barcode databases.Panel (A) Sanger-generated barcodes. Panel (B) Illumina-generated barcodes.

Mentions: All sequences produced by both Sanger and Illumina MiSeq sequencing were identified via BLAST28 comparison to public COI databases. Each top hit BLAST result for each sequence for each individual was then compared to morphological identification (Fig. 2). A total of 509 individuals (50.4%) produced a DNA sequence matching morphological identification via Sanger sequencing. The number of non-matching sequences was 28 (2.8%), with the remaining 473 individuals (46.8%) producing no Sanger sequence at all. The percentage of matching Sanger sequences differed between arthropod orders, ranging from 1.8% for Trombidiformes to 62.4% for Hymenoptera, 63.2% for Diptera and 87.5% for Lepidoptera (Fig. 2A).


Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform.

Shokralla S, Porter TM, Gibson JF, Dobosz R, Janzen DH, Hallwachs W, Golding GB, Hajibabaei M - Sci Rep (2015)

Number and percentage of 1,010 individual arthropod specimens producing a COI DNA sequence that matches morphological identification based on BLAST comparison to public DNA barcode databases.Panel (A) Sanger-generated barcodes. Panel (B) Illumina-generated barcodes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401116&req=5

f2: Number and percentage of 1,010 individual arthropod specimens producing a COI DNA sequence that matches morphological identification based on BLAST comparison to public DNA barcode databases.Panel (A) Sanger-generated barcodes. Panel (B) Illumina-generated barcodes.
Mentions: All sequences produced by both Sanger and Illumina MiSeq sequencing were identified via BLAST28 comparison to public COI databases. Each top hit BLAST result for each sequence for each individual was then compared to morphological identification (Fig. 2). A total of 509 individuals (50.4%) produced a DNA sequence matching morphological identification via Sanger sequencing. The number of non-matching sequences was 28 (2.8%), with the remaining 473 individuals (46.8%) producing no Sanger sequence at all. The percentage of matching Sanger sequences differed between arthropod orders, ranging from 1.8% for Trombidiformes to 62.4% for Hymenoptera, 63.2% for Diptera and 87.5% for Lepidoptera (Fig. 2A).

Bottom Line: Here, we present a scalable double dual-indexing approach using an Illumina Miseq platform to sequence DNA barcode markers.We achieved 97.3% success by using half of an Illumina Miseq flowcell to obtain 658 base pairs of the cytochrome c oxidase I DNA barcode in 1,010 specimens from eleven orders of arthropods.Our approach recovers a greater proportion of DNA barcode sequences from individuals than does conventional Sanger sequencing, while at the same time reducing both per specimen costs and labor time by nearly 80%.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON, Canada N1G 2W1.

ABSTRACT
Genetic information is a valuable component of biosystematics, especially specimen identification through the use of species-specific DNA barcodes. Although many genomics applications have shifted to High-Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies, sample identification (e.g., via DNA barcoding) is still most often done with Sanger sequencing. Here, we present a scalable double dual-indexing approach using an Illumina Miseq platform to sequence DNA barcode markers. We achieved 97.3% success by using half of an Illumina Miseq flowcell to obtain 658 base pairs of the cytochrome c oxidase I DNA barcode in 1,010 specimens from eleven orders of arthropods. Our approach recovers a greater proportion of DNA barcode sequences from individuals than does conventional Sanger sequencing, while at the same time reducing both per specimen costs and labor time by nearly 80%. In addition, the use of HTS allows the recovery of multiple sequences per specimen, for deeper analysis of genetic variation in target gene regions.

No MeSH data available.