Dataset Information

TGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci.

ABSTRACT: Conventional genotyping-by-sequencing (cGBS) strategies suffer from high rates of missing data and genotyping errors, particularly at heterozygous sites. tGBS® genotyping-by-sequencing is a novel method of genome reduction that employs two restriction enzymes to generate overhangs in opposite orientations to which (single-strand) oligos rather than (double-stranded) adaptors are ligated. This strategy ensures that only double-digested fragments are amplified and sequenced. The use of oligos avoids the necessity of preparing adaptors and the problems associated with inter-adaptor annealing/ligation. Hence, the tGBS protocol simplifies the preparation of high-quality GBS sequencing libraries. During polymerase chain reaction (PCR) amplification, selective nucleotides included at the 3'-end of the PCR primers result in additional genome reduction as compared to cGBS. By adjusting the number of selective bases, different numbers of genomic sites are targeted for sequencing. Therefore, for equivalent amounts of sequencing, more reads per site are available for SNP calling. Hence, as compared to cGBS, tGBS delivers higher SNP calling accuracy (>97-99%), even at heterozygous sites, less missing data per marker across a population of samples, and an enhanced ability to genotype rare alleles. tGBS is particularly well suited for genomic selection, which often requires the ability to genotype populations of individuals that are heterozygous at many loci.

SUBMITTER: Ott A

PROVIDER: S-EPMC5716196 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci.

Ott Alina A Liu Sanzhen S Schnable James C JC Yeh Cheng-Ting 'Eddy' C' Wang Kai-Sin KS Schnable Patrick S PS

Nucleic acids research 20171201 21

Conventional genotyping-by-sequencing (cGBS) strategies suffer from high rates of missing data and genotyping errors, particularly at heterozygous sites. tGBS® genotyping-by-sequencing is a novel method of genome reduction that employs two restriction enzymes to generate overhangs in opposite orientations to which (single-strand) oligos rather than (double-stranded) adaptors are ligated. This strategy ensures that only double-digested fragments are amplified and sequenced. The use of oligos avoi ...[more]

PMID: 29036322

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Project description:Assessment of genomic DNA sequence variation and genotype calling in autotetraploids implies the ability to distinguish among five possible alternative allele copy number states. This study demonstrates the accuracy of genotyping-by-sequencing (GBS) of a large collection of autotetraploid potato cultivars using next-generation sequencing. It is still costly to reach sufficient read depths on a genome wide scale, across the cultivated gene pool. Therefore, we enriched cultivar-specific DNA sequencing libraries using an in-solution hybridisation method (SureSelect). This complexity reduction allowed to confine our study to 807 target genes distributed across the genomes of 83 tetraploid cultivars and one reference (DM 1-3 511). Indexed sequencing libraries were paired-end sequenced in 7 pools of 12 samples using Illumina HiSeq2000. After filtering and processing the raw sequence data, 12.4 Gigabases of high-quality sequence data was obtained, which mapped to 2.1 Mb of the potato reference genome, with a median average read depth of 63× per cultivar. We detected 129,156 sequence variants and genotyped the allele copy number of each variant for every cultivar. In this cultivar panel a variant density of 1 SNP/24 bp in exons and 1 SNP/15 bp in introns was obtained. The average minor allele frequency (MAF) of a variant was 0.14. Potato germplasm displayed a large number of relatively rare variants and/or haplotypes, with 61% of the variants having a MAF below 0.05. A very high average nucleotide diversity (??=?0.0107) was observed. Nucleotide diversity varied among potato chromosomes. Several genes under selection were identified. Genotyping-by-sequencing results, with allele copy number estimates, were validated with a KASP genotyping assay. This validation showed that read depths of ?60-80× can be used as a lower boundary for reliable assessment of allele copy number of sequence variants in autotetraploids. Genotypic data were associated with traits, and alleles strongly influencing maturity and flesh colour were identified.

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Dataset Information

TGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci.

Publications

tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci.

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