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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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Linkage groups incorporating newly-developed transcriptome SNP markers. Transcriptome SNP markers assayed by the BAT method mapped to the anticipated linkage groups (A1, A5, C1, C5 and C3) showing and are shown in red.
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Figure 3: Linkage groups incorporating newly-developed transcriptome SNP markers. Transcriptome SNP markers assayed by the BAT method mapped to the anticipated linkage groups (A1, A5, C1, C5 and C3) showing and are shown in red.

Mentions: The resulting scoring strings for 24 markers (derived from 17 amplicons) were successfully linkage mapped, as shown in FigureĀ 3 and listed in Additional file3, to the expected regions of the genome, i.e. 14 markers on linkage groups A3, A5, C1 and C5, and 10 markers on linkage groups A1, A3 and A5. The remaining SNPs could not be mapped due to lack of polymorphism or because read depth was too low for robust allele-calling. For validation, Illumina GoldenGate platform[11] markers were developed for eight of the unigenes containing SNPs mapped by the BAT method and scored across the population. Also, the scoring strings from the published oilseed rape SNP map[17] for these eight unigenes were retrieved, and the scoring strings for both types of markers compared with those obtained by the BAT method, as shown in Additional file4. The BAT markers showed a high accuracy of scoring, with only 5 mis-calls in the 483 alleles scored (1.0%).


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)

Linkage groups incorporating newly-developed transcriptome SNP markers. Transcriptome SNP markers assayed by the BAT method mapped to the anticipated linkage groups (A1, A5, C1, C5 and C3) showing and are shown in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Linkage groups incorporating newly-developed transcriptome SNP markers. Transcriptome SNP markers assayed by the BAT method mapped to the anticipated linkage groups (A1, A5, C1, C5 and C3) showing and are shown in red.
Mentions: The resulting scoring strings for 24 markers (derived from 17 amplicons) were successfully linkage mapped, as shown in FigureĀ 3 and listed in Additional file3, to the expected regions of the genome, i.e. 14 markers on linkage groups A3, A5, C1 and C5, and 10 markers on linkage groups A1, A3 and A5. The remaining SNPs could not be mapped due to lack of polymorphism or because read depth was too low for robust allele-calling. For validation, Illumina GoldenGate platform[11] markers were developed for eight of the unigenes containing SNPs mapped by the BAT method and scored across the population. Also, the scoring strings from the published oilseed rape SNP map[17] for these eight unigenes were retrieved, and the scoring strings for both types of markers compared with those obtained by the BAT method, as shown in Additional file4. The BAT markers showed a high accuracy of scoring, with only 5 mis-calls in the 483 alleles scored (1.0%).

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