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High-throughput detection of induced mutations and natural variation using KeyPoint technology.

Rigola D, van Oeveren J, Janssen A, Bonné A, Schneiders H, van der Poel HJ, van Orsouw NJ, Hogers RC, de Both MT, van Eijk MJ - PLoS ONE (2009)

Bottom Line: We present KeyPoint technology, a high-throughput mutation/polymorphism discovery technique based on massive parallel sequencing of target genes amplified from mutant or natural populations.We show the power of KeyPoint by identifying two mutants in the tomato eIF4E gene based on screening more than 3000 M2 families in a single GS FLX sequencing run, and discovery of six haplotypes of tomato eIF4E gene by re-sequencing three amplicons in a subset of 92 tomato lines from the EU-SOL core collection.We propose KeyPoint technology as a broadly applicable amplicon sequencing approach to screen mutant populations or germplasm collections for identification of (novel) allelic variation in a high-throughput fashion.

View Article: PubMed Central - PubMed

Affiliation: Keygene NV, Wageningen, The Netherlands. diana.rigola@keygene.com

ABSTRACT
Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variants among mutant or natural populations. Current (pre-) screening methods such as TILLING and EcoTILLING are based on the detection of single base mismatches in heteroduplexes using endonucleases such as CEL 1. However, there are drawbacks in the use of endonucleases due to their relatively poor cleavage efficiency and exonuclease activity. Moreover, pre-screening methods do not reveal information about the nature of sequence changes and their possible impact on gene function. We present KeyPoint technology, a high-throughput mutation/polymorphism discovery technique based on massive parallel sequencing of target genes amplified from mutant or natural populations. KeyPoint combines multi-dimensional pooling of large numbers of individual DNA samples and the use of sample identification tags ("sample barcoding") with next-generation sequencing technology. We show the power of KeyPoint by identifying two mutants in the tomato eIF4E gene based on screening more than 3000 M2 families in a single GS FLX sequencing run, and discovery of six haplotypes of tomato eIF4E gene by re-sequencing three amplicons in a subset of 92 tomato lines from the EU-SOL core collection. We propose KeyPoint technology as a broadly applicable amplicon sequencing approach to screen mutant populations or germplasm collections for identification of (novel) allelic variation in a high-throughput fashion.

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KeyPoint flowchart.
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pone-0004761-g001: KeyPoint flowchart.

Mentions: The recent introduction of instruments capable of producing millions of DNA sequence reads in a single experiment opened the possibility of developing a new high-throughput mutation discovery technology based on massive parallel sequencing. Here we describe the KeyPoint™ technology, a novel mutation/polymorphism screening technique using the Genome Sequencer (GS) FLX platform (Roche Applied Science), which allows massive parallel picoliter-scale amplification and pyrosequencing of individual DNA molecules [20]. Using KeyPoint, genes of interest are directly amplified by PCR and sequenced. To significantly reduce sample preparation costs, KeyPoint applies a multidimensional pooling strategy of amplification templates (DNA samples) belonging to mutant or natural populations. Gene-specific PCR primers carry sample identification tags specific for each multidimensional pool in order to assign sequence reads to individual mutant plants or to assign sequence haplotypes to pooled or individual samples of a germplasm collection (Fig. 1). Using custom developed bio-informatics tools the sequence reads are clustered, aligned, and mined for mutations or single nucleotide polymorphisms (SNPs). Statistical probability calculation methods are used to distinguish true mutations and polymorphisms from amplification or sequencing errors. With the KeyPoint technology we identify two EMS induced mutations in exon 1 of the tomato (Solanum lycopersicum) eukaryotic translation initiation factor 4E (SleIF4E) gene by screening 15,040 mutant tomato plants (five segregating M2 plants of each of 3008 M2 families) in a single GS FLX run. In addition, power calculations were performed to define the throughput of the KeyPoint technology. Finally, KeyPoint revealed at least six naturally occurring haplotypes defined by fifteen SNPs observed in three amplicons of the SleIF4E gene in a subset of 92 lines of the EU-SOL tomato germplasm core collection in just 1/4 GS FLX run.


High-throughput detection of induced mutations and natural variation using KeyPoint technology.

Rigola D, van Oeveren J, Janssen A, Bonné A, Schneiders H, van der Poel HJ, van Orsouw NJ, Hogers RC, de Both MT, van Eijk MJ - PLoS ONE (2009)

KeyPoint flowchart.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654077&req=5

pone-0004761-g001: KeyPoint flowchart.
Mentions: The recent introduction of instruments capable of producing millions of DNA sequence reads in a single experiment opened the possibility of developing a new high-throughput mutation discovery technology based on massive parallel sequencing. Here we describe the KeyPoint™ technology, a novel mutation/polymorphism screening technique using the Genome Sequencer (GS) FLX platform (Roche Applied Science), which allows massive parallel picoliter-scale amplification and pyrosequencing of individual DNA molecules [20]. Using KeyPoint, genes of interest are directly amplified by PCR and sequenced. To significantly reduce sample preparation costs, KeyPoint applies a multidimensional pooling strategy of amplification templates (DNA samples) belonging to mutant or natural populations. Gene-specific PCR primers carry sample identification tags specific for each multidimensional pool in order to assign sequence reads to individual mutant plants or to assign sequence haplotypes to pooled or individual samples of a germplasm collection (Fig. 1). Using custom developed bio-informatics tools the sequence reads are clustered, aligned, and mined for mutations or single nucleotide polymorphisms (SNPs). Statistical probability calculation methods are used to distinguish true mutations and polymorphisms from amplification or sequencing errors. With the KeyPoint technology we identify two EMS induced mutations in exon 1 of the tomato (Solanum lycopersicum) eukaryotic translation initiation factor 4E (SleIF4E) gene by screening 15,040 mutant tomato plants (five segregating M2 plants of each of 3008 M2 families) in a single GS FLX run. In addition, power calculations were performed to define the throughput of the KeyPoint technology. Finally, KeyPoint revealed at least six naturally occurring haplotypes defined by fifteen SNPs observed in three amplicons of the SleIF4E gene in a subset of 92 lines of the EU-SOL tomato germplasm core collection in just 1/4 GS FLX run.

Bottom Line: We present KeyPoint technology, a high-throughput mutation/polymorphism discovery technique based on massive parallel sequencing of target genes amplified from mutant or natural populations.We show the power of KeyPoint by identifying two mutants in the tomato eIF4E gene based on screening more than 3000 M2 families in a single GS FLX sequencing run, and discovery of six haplotypes of tomato eIF4E gene by re-sequencing three amplicons in a subset of 92 tomato lines from the EU-SOL core collection.We propose KeyPoint technology as a broadly applicable amplicon sequencing approach to screen mutant populations or germplasm collections for identification of (novel) allelic variation in a high-throughput fashion.

View Article: PubMed Central - PubMed

Affiliation: Keygene NV, Wageningen, The Netherlands. diana.rigola@keygene.com

ABSTRACT
Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variants among mutant or natural populations. Current (pre-) screening methods such as TILLING and EcoTILLING are based on the detection of single base mismatches in heteroduplexes using endonucleases such as CEL 1. However, there are drawbacks in the use of endonucleases due to their relatively poor cleavage efficiency and exonuclease activity. Moreover, pre-screening methods do not reveal information about the nature of sequence changes and their possible impact on gene function. We present KeyPoint technology, a high-throughput mutation/polymorphism discovery technique based on massive parallel sequencing of target genes amplified from mutant or natural populations. KeyPoint combines multi-dimensional pooling of large numbers of individual DNA samples and the use of sample identification tags ("sample barcoding") with next-generation sequencing technology. We show the power of KeyPoint by identifying two mutants in the tomato eIF4E gene based on screening more than 3000 M2 families in a single GS FLX sequencing run, and discovery of six haplotypes of tomato eIF4E gene by re-sequencing three amplicons in a subset of 92 tomato lines from the EU-SOL core collection. We propose KeyPoint technology as a broadly applicable amplicon sequencing approach to screen mutant populations or germplasm collections for identification of (novel) allelic variation in a high-throughput fashion.

Show MeSH
Related in: MedlinePlus