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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.


Neighbor-joining diagram of 1,211 COI sequences produced from Illumina MiSeq sequencing of 1,010 individual arthropods.Distance measurement is calculated in number of base substitutions per site based on the Kimura 2-parameter method. Sequences originating from individuals morphologically identified as Coleoptera (blue), Psocoptera (red), and Trombidiformes (green) are indicated. Distinction is also made between sequences that correctly matched morphology based on a BLAST comparison to public COI databases (outlined), and those that did not match morphology (filled in). Individuals producing a single sequence are depicted as circles, whereas multiple sequences from the same individuals are depicted with triangles.
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f3: Neighbor-joining diagram of 1,211 COI sequences produced from Illumina MiSeq sequencing of 1,010 individual arthropods.Distance measurement is calculated in number of base substitutions per site based on the Kimura 2-parameter method. Sequences originating from individuals morphologically identified as Coleoptera (blue), Psocoptera (red), and Trombidiformes (green) are indicated. Distinction is also made between sequences that correctly matched morphology based on a BLAST comparison to public COI databases (outlined), and those that did not match morphology (filled in). Individuals producing a single sequence are depicted as circles, whereas multiple sequences from the same individuals are depicted with triangles.

Mentions: Individuals from the three arthropod orders with the lowest percentage of matching Illumina MiSeq sequences to morphology were selected for further analysis. Coleoptera, Psocoptera, and Trombidiformes had the highest percentages of non-matching Illumina MiSeq sequences when compared to the morphological identification (38.3%, 72.9%, and 98.2% respectively) (Fig. 2). All unique sequences produced by Illumina MiSeq (n = 1,211) were used for a Neighbor-Joining analysis based on pairwise distance (Fig. 3). Sequences recovered from Coleoptera, Psocoptera, and Trombidiformes were labeled as either matching or non-matching. Distinction was also made between individuals producing a single Illumina MiSeq sequence and individuals producing multiple sequences. For sequences recovered from individuals identified morphologically as Coleoptera, all but eight were contained within a single cluster including all matching sequences. The same case was true for Psocoptera, with only six sequences excluded, and Trombidiformes, with only one sequence excluded.


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)

Neighbor-joining diagram of 1,211 COI sequences produced from Illumina MiSeq sequencing of 1,010 individual arthropods.Distance measurement is calculated in number of base substitutions per site based on the Kimura 2-parameter method. Sequences originating from individuals morphologically identified as Coleoptera (blue), Psocoptera (red), and Trombidiformes (green) are indicated. Distinction is also made between sequences that correctly matched morphology based on a BLAST comparison to public COI databases (outlined), and those that did not match morphology (filled in). Individuals producing a single sequence are depicted as circles, whereas multiple sequences from the same individuals are depicted with triangles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Neighbor-joining diagram of 1,211 COI sequences produced from Illumina MiSeq sequencing of 1,010 individual arthropods.Distance measurement is calculated in number of base substitutions per site based on the Kimura 2-parameter method. Sequences originating from individuals morphologically identified as Coleoptera (blue), Psocoptera (red), and Trombidiformes (green) are indicated. Distinction is also made between sequences that correctly matched morphology based on a BLAST comparison to public COI databases (outlined), and those that did not match morphology (filled in). Individuals producing a single sequence are depicted as circles, whereas multiple sequences from the same individuals are depicted with triangles.
Mentions: Individuals from the three arthropod orders with the lowest percentage of matching Illumina MiSeq sequences to morphology were selected for further analysis. Coleoptera, Psocoptera, and Trombidiformes had the highest percentages of non-matching Illumina MiSeq sequences when compared to the morphological identification (38.3%, 72.9%, and 98.2% respectively) (Fig. 2). All unique sequences produced by Illumina MiSeq (n = 1,211) were used for a Neighbor-Joining analysis based on pairwise distance (Fig. 3). Sequences recovered from Coleoptera, Psocoptera, and Trombidiformes were labeled as either matching or non-matching. Distinction was also made between individuals producing a single Illumina MiSeq sequence and individuals producing multiple sequences. For sequences recovered from individuals identified morphologically as Coleoptera, all but eight were contained within a single cluster including all matching sequences. The same case was true for Psocoptera, with only six sequences excluded, and Trombidiformes, with only one sequence excluded.

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.