Limits...
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.


Workflow and cost and time analysis for the generation of DNA sequence data from multiple specimens using Illumina MiSeq sequencing.(A) The recommended new workflow. (B) A cost and time analysis of the new workflow versus conventional Sanger sequencing for ~1000 individuals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4401116&req=5

f5: Workflow and cost and time analysis for the generation of DNA sequence data from multiple specimens using Illumina MiSeq sequencing.(A) The recommended new workflow. (B) A cost and time analysis of the new workflow versus conventional Sanger sequencing for ~1000 individuals.

Mentions: We recommend a new workflow for generating DNA barcode sequences (Fig. 5A). Morphological identification of specimens is optional within the workflow and could be completed at a later time for confirmatory purposes. The method is adaptable to all organisms (i.e., plants, animals, fungi, bacteria) and all genetic markers (i.e., COI, ITS, rbcL, 16S, 18S). We calculated the cost and time investments in DNA sequence generation using Sanger sequencing compared to our new method (Fig. 5B). The new method represents a 27% reduction in total time and 78% reduction in hands-on time in addition to a 79% reduction in laboratory costs. This cost reduction will increase with projected advances in HTS technology. The presented workflow also allows research laboratories to employ a single HTS platform for both metabarcoding of bulk environmental samples and the generation of barcodes for individual specimens.


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)

Workflow and cost and time analysis for the generation of DNA sequence data from multiple specimens using Illumina MiSeq sequencing.(A) The recommended new workflow. (B) A cost and time analysis of the new workflow versus conventional Sanger sequencing for ~1000 individuals.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Workflow and cost and time analysis for the generation of DNA sequence data from multiple specimens using Illumina MiSeq sequencing.(A) The recommended new workflow. (B) A cost and time analysis of the new workflow versus conventional Sanger sequencing for ~1000 individuals.
Mentions: We recommend a new workflow for generating DNA barcode sequences (Fig. 5A). Morphological identification of specimens is optional within the workflow and could be completed at a later time for confirmatory purposes. The method is adaptable to all organisms (i.e., plants, animals, fungi, bacteria) and all genetic markers (i.e., COI, ITS, rbcL, 16S, 18S). We calculated the cost and time investments in DNA sequence generation using Sanger sequencing compared to our new method (Fig. 5B). The new method represents a 27% reduction in total time and 78% reduction in hands-on time in addition to a 79% reduction in laboratory costs. This cost reduction will increase with projected advances in HTS technology. The presented workflow also allows research laboratories to employ a single HTS platform for both metabarcoding of bulk environmental samples and the generation of barcodes for individual specimens.

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.