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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.


Pairwise distances between COI DNA sequences generated by Sanger-sequencing and Illumina MiSeq sequencing for 521 individual arthropods.Circles represent the first Illumina generated cluster, most similar to the Sanger, with other symbols representing second, third, and fourth Illumina sequences generated from the same individual. The area below the dashed line represents all Illumina sequences sharing at least 98% sequence similarity with a corresponding Sanger sequence from the same individual.
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f4: Pairwise distances between COI DNA sequences generated by Sanger-sequencing and Illumina MiSeq sequencing for 521 individual arthropods.Circles represent the first Illumina generated cluster, most similar to the Sanger, with other symbols representing second, third, and fourth Illumina sequences generated from the same individual. The area below the dashed line represents all Illumina sequences sharing at least 98% sequence similarity with a corresponding Sanger sequence from the same individual.

Mentions: To investigate the accuracy of the Illumina barcoding approach as compared to Sanger sequencing, pairwise distances between Sanger and Illumina sequences generated by the same individual were calculated (Fig. 4). Of the 521 individuals for which both Illumina and Sanger sequences were produced, 429 (82%) produced Sanger and Illumina sequences with no sequence difference. A total of 463 (89%) individuals produced Sanger and Illumina sequences with less than 2% sequence difference.


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)

Pairwise distances between COI DNA sequences generated by Sanger-sequencing and Illumina MiSeq sequencing for 521 individual arthropods.Circles represent the first Illumina generated cluster, most similar to the Sanger, with other symbols representing second, third, and fourth Illumina sequences generated from the same individual. The area below the dashed line represents all Illumina sequences sharing at least 98% sequence similarity with a corresponding Sanger sequence from the same individual.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Pairwise distances between COI DNA sequences generated by Sanger-sequencing and Illumina MiSeq sequencing for 521 individual arthropods.Circles represent the first Illumina generated cluster, most similar to the Sanger, with other symbols representing second, third, and fourth Illumina sequences generated from the same individual. The area below the dashed line represents all Illumina sequences sharing at least 98% sequence similarity with a corresponding Sanger sequence from the same individual.
Mentions: To investigate the accuracy of the Illumina barcoding approach as compared to Sanger sequencing, pairwise distances between Sanger and Illumina sequences generated by the same individual were calculated (Fig. 4). Of the 521 individuals for which both Illumina and Sanger sequences were produced, 429 (82%) produced Sanger and Illumina sequences with no sequence difference. A total of 463 (89%) individuals produced Sanger and Illumina sequences with less than 2% sequence difference.

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.