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Sequencing-based variant detection in the polyploid crop oilseed rape.

Wells R, Trick M, Fraser F, Soumpourou E, Clissold L, Morgan C, Pauquet J, Bancroft I - BMC Plant Biol. (2013)

Bottom Line: Using this methodology, amplicons targeted to gene sequences were screened across a B. napus mapping population and the resulting allele scoring strings for 24 markers linkage mapped to the expected regions of the genome.The utilisation of barcode tags to de-convolute pooled PCR products in multiplexed, variation screening via Illumina sequencing provides a cost effective method for SNP genotyping and mutation detection and, potentially, markers for causative changes, even in polyploid species.Combining this approach with existing Illumina multiplexing workflows allows the analysis of thousands of lines cheaply and efficiently in a single sequencing run with minimal library production costs.

View Article: PubMed Central - HTML - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

ABSTRACT

Background: The detection and exploitation of genetic variation underpins crop improvement. However, the polyploid nature of the genomes of many of our most important crops represents a barrier, particularly for the analysis of variation within genes. To overcome this, we aimed to develop methodologies based on amplicon sequencing that involve the incorporation of barcoded amplification tags (BATs) into PCR products.

Results: A protocol was developed to tag PCR products with 5' 6-base oligonucleotide barcode extensions before pooling for sequencing library production using standard Illumina adapters. A computational method was developed for the de-convolution of products and the robust detection and scoring of sequence variants. Using this methodology, amplicons targeted to gene sequences were screened across a B. napus mapping population and the resulting allele scoring strings for 24 markers linkage mapped to the expected regions of the genome. Furthermore, using one-dimensional 8-fold pooling, 4608 lines of a B. napus mutation population were screened for induced mutations in a locus-specific amplicon (an orthologue of GL2.b) and mixed product of three co-amplified loci (orthologues of FAD2), identifying 10 and 41 mutants respectively.

Conclusions: The utilisation of barcode tags to de-convolute pooled PCR products in multiplexed, variation screening via Illumina sequencing provides a cost effective method for SNP genotyping and mutation detection and, potentially, markers for causative changes, even in polyploid species. Combining this approach with existing Illumina multiplexing workflows allows the analysis of thousands of lines cheaply and efficiently in a single sequencing run with minimal library production costs.

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Schematic showing the addition of the barcode tag during PCR amplification. In the initial cycles, M13(−21) tails are incorporated into the PCR product however due to the limited amount of specific M13(−21) primer this is replaced with the labelling with the barcode in subsequent cycles.
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Figure 1: Schematic showing the addition of the barcode tag during PCR amplification. In the initial cycles, M13(−21) tails are incorporated into the PCR product however due to the limited amount of specific M13(−21) primer this is replaced with the labelling with the barcode in subsequent cycles.

Mentions: An existing method for the addition of fluorescent labels to PCR products[8] was used as the basis for our method for the addition of oligonucleotide barcodes to PCR products. Three oligonucleotides are used in the PCR reactions: (1) a sequence-specific forward primer with M13(−21) tail at its 5’ end, (2) a universal M13(−21) primer with oligonucleotide barcode tail at its 5’ end (hereafter the barcoded amplification tag; BAT) and (3) a sequence-specific reverse primer. By limiting the amount in the reaction of the sequence-specific forward primer with M13(−21) tail, PCR can be conducted in a single-stage reaction in which early rounds of amplification incorporate the universal forward primer and later rounds incorporate the BAT primer. The barcoding process is shown schematically in Figure 1, with the BAT experimental design illustrated schematically in Figure 2.


Sequencing-based variant detection in the polyploid crop oilseed rape.

Wells R, Trick M, Fraser F, Soumpourou E, Clissold L, Morgan C, Pauquet J, Bancroft I - BMC Plant Biol. (2013)

Schematic showing the addition of the barcode tag during PCR amplification. In the initial cycles, M13(−21) tails are incorporated into the PCR product however due to the limited amount of specific M13(−21) primer this is replaced with the labelling with the barcode in subsequent cycles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750413&req=5

Figure 1: Schematic showing the addition of the barcode tag during PCR amplification. In the initial cycles, M13(−21) tails are incorporated into the PCR product however due to the limited amount of specific M13(−21) primer this is replaced with the labelling with the barcode in subsequent cycles.
Mentions: An existing method for the addition of fluorescent labels to PCR products[8] was used as the basis for our method for the addition of oligonucleotide barcodes to PCR products. Three oligonucleotides are used in the PCR reactions: (1) a sequence-specific forward primer with M13(−21) tail at its 5’ end, (2) a universal M13(−21) primer with oligonucleotide barcode tail at its 5’ end (hereafter the barcoded amplification tag; BAT) and (3) a sequence-specific reverse primer. By limiting the amount in the reaction of the sequence-specific forward primer with M13(−21) tail, PCR can be conducted in a single-stage reaction in which early rounds of amplification incorporate the universal forward primer and later rounds incorporate the BAT primer. The barcoding process is shown schematically in Figure 1, with the BAT experimental design illustrated schematically in Figure 2.

Bottom Line: Using this methodology, amplicons targeted to gene sequences were screened across a B. napus mapping population and the resulting allele scoring strings for 24 markers linkage mapped to the expected regions of the genome.The utilisation of barcode tags to de-convolute pooled PCR products in multiplexed, variation screening via Illumina sequencing provides a cost effective method for SNP genotyping and mutation detection and, potentially, markers for causative changes, even in polyploid species.Combining this approach with existing Illumina multiplexing workflows allows the analysis of thousands of lines cheaply and efficiently in a single sequencing run with minimal library production costs.

View Article: PubMed Central - HTML - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

ABSTRACT

Background: The detection and exploitation of genetic variation underpins crop improvement. However, the polyploid nature of the genomes of many of our most important crops represents a barrier, particularly for the analysis of variation within genes. To overcome this, we aimed to develop methodologies based on amplicon sequencing that involve the incorporation of barcoded amplification tags (BATs) into PCR products.

Results: A protocol was developed to tag PCR products with 5' 6-base oligonucleotide barcode extensions before pooling for sequencing library production using standard Illumina adapters. A computational method was developed for the de-convolution of products and the robust detection and scoring of sequence variants. Using this methodology, amplicons targeted to gene sequences were screened across a B. napus mapping population and the resulting allele scoring strings for 24 markers linkage mapped to the expected regions of the genome. Furthermore, using one-dimensional 8-fold pooling, 4608 lines of a B. napus mutation population were screened for induced mutations in a locus-specific amplicon (an orthologue of GL2.b) and mixed product of three co-amplified loci (orthologues of FAD2), identifying 10 and 41 mutants respectively.

Conclusions: The utilisation of barcode tags to de-convolute pooled PCR products in multiplexed, variation screening via Illumina sequencing provides a cost effective method for SNP genotyping and mutation detection and, potentially, markers for causative changes, even in polyploid species. Combining this approach with existing Illumina multiplexing workflows allows the analysis of thousands of lines cheaply and efficiently in a single sequencing run with minimal library production costs.

Show MeSH
Related in: MedlinePlus