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Genotyping and haplotyping of polymorphisms directly from genomic DNA via coupled amplification and sequencing (CAS).

ABSTRACT: Coupled amplification and sequencing (CAS) allows a segment of DNA to be sequenced directly from genomic DNA. An initial PCR amplification stage selects and amplifies the target. During a subsequent stage both strands of the target segment are sequenced simultaneously and amplified further. We show that CAS can readily identify variant base pairs. Genotyping of a population for known sequence variation can be achieved simply and directly from genomic DNA of each organism by performing CAS only for the variant bases. The procedure supercedes development and optimization of alternative typing assays based on oligonucleotide hybridization or ligation. In addition, we show that competitive oligonucleotide priming with allelic primers can be readily performed in concert with the second stage of CAS. The combination of techniques allows sequencing of a single chromosome from a heterozygous genomic sample and direct haplotyping of the polymorphism at the priming site with any others encompassed within the amplified segment.

SUBMITTER: Ruano G

PROVIDER: S-EPMC329323 | biostudies-other | 1991 Dec

REPOSITORIES: biostudies-other

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Genotyping and haplotyping of polymorphisms directly from genomic DNA via coupled amplification and sequencing (CAS).

Ruano G G Kidd K K KK

Nucleic acids research 19911201 24

Coupled amplification and sequencing (CAS) allows a segment of DNA to be sequenced directly from genomic DNA. An initial PCR amplification stage selects and amplifies the target. During a subsequent stage both strands of the target segment are sequenced simultaneously and amplified further. We show that CAS can readily identify variant base pairs. Genotyping of a population for known sequence variation can be achieved simply and directly from genomic DNA of each organism by performing CAS only f ...[more]

PMID: 1684844

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Project description:Enriching of kernel zinc (Zn) concentration in maize is one of the most effective ways to solve the problem of Zn deficiency in low and middle income countries where maize is the major staple food, and 17% of the global population is affected with Zn deficiency. Genomic selection (GS) has shown to be an effective approach to accelerate genetic gains in plant breeding. In the present study, an association-mapping panel and two maize double-haploid (DH) populations, both genotyped with genotyping-by-sequencing (GBS) and repeat amplification sequencing (rAmpSeq) markers, were used to estimate the genomic prediction accuracy of kernel Zn concentration in maize. Results showed that the prediction accuracy of two DH populations was higher than that of the association mapping population using the same set of markers. The prediction accuracy estimated with the GBS markers was significantly higher than that estimated with the rAmpSeq markers in the same population. The maximum prediction accuracy with minimum standard error was observed when half of the genotypes were included in the training set and 3,000 and 500 markers were used for prediction in the association mapping panel and the DH populations, respectively. Appropriate levels of minor allele frequency and missing rate should be considered and selected to achieve good prediction accuracy and reduce the computation burden by balancing the number of markers and marker quality. Training set development with broad phenotypic variation is possible to improve prediction accuracy. The transferability of the GS models across populations was assessed, the prediction accuracies in a few pairwise populations were above or close to 0.20, which indicates the prediction accuracies across years and populations have to be assessed in a larger breeding dataset with closer relationship between the training and prediction sets in further studies. GS outperformed MAS (marker-assisted-selection) on predicting the kernel Zn concentration in maize, the decision of a breeding strategy to implement GS individually or to implement MAS and GS stepwise for improving kernel Zn concentration in maize requires further research. Results of this study provide valuable information for understanding how to implement GS for improving kernel Zn concentration in maize.

Project description:BackgroundThe recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or "orphan" crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6-$9/sample) for consumables.ResultsWe modified a non-organic solvent protocol to extract microgram quantities (1-13 μg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384-576 samples/day), and is inexpensive at US$0.62/sample for consumables.ConclusionsThis versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

Dataset Information

Genotyping and haplotyping of polymorphisms directly from genomic DNA via coupled amplification and sequencing (CAS).

Publications

Genotyping and haplotyping of polymorphisms directly from genomic DNA via coupled amplification and sequencing (CAS).

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