GRAS-Di® for Marker discovery & Genotyping⚫ GRAS-Di® (Genotyping by Random Amplicon Sequencing-Direct) was developed
by TOYOTA Motor Corporation⚫ GRAS-Di® amplifies the random regions throughout the genome for NextGeneration
Sequencing.
⚫ Skim based sequencing of whole genome covered by the amplicons through 2 step
PCR with random primers, Non-targeted PCR-based GBS.
⚫ Sequencing data is analyzed for presence / absence of amplicon markers andoptionally SNPs calling.

GRAS-Di® vs Whole Genome Sequence(WGS)⚫ GRAS-Di® : Sequence reads are uniformly mapped to the genome at intervals.
⚫ WGS:

Sequence reads are uniformly mapped to the genome without gaps.

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Amplicons covering on the chromosomes⚫ Mapping of Amplicons onto the reference sequence shows the uniform
distribution of Markers over the genome

4GRAS-Di® - Advantages⚫ More cost effective than Whole Genome deep Sequencing⚫ Economical & Ecological, simple method that tolerates low quality DNA or slightly
fragmented DNA (>100ng)⚫ Applicable to both natural and segregated populations, all without the need for areference genome⚫ Obtains fewer missing data than RAD-seq

GRAS-Di® - Applications
Applicable for Genetic diversity studies, Polymorphism detections (SNPs or InDels or Haplotypes).
Increased usage in the field of Phylogenetic and population structure studies.

SNP Discovery⚫ Develop SNP markers for translation to a preferred genotyping system⚫ High quality alternative to GBS/WGS, RNA-Seq, RAD-Seq, ddRAD-Seq (GBS)High Throughput Genotyping⚫ Discover and genotype massive amounts of genetic variants for Marker Selection⚫ High-density Linkage Mapping & QTL Mapping, Chromosome Mapping, Phylogenic &Population structure studies

Others⚫ Potential of large number of polymorphisms used as molecular markers for genetic analysis⚫ Possible to obtain genome-wide co-dominant markers Identify many markers covering all
chromosomes even in a population with small genetic variation

GRAS-Di® Analysis Service - Experiences
GRAS-Di® has been implemented successfully in over 120 different
species including crops, forestry, livestock and aquaculture.
The technology is also applicable to highly polyploid species.
Please refer the Bibliography for further details.

Agriculture/Plant

Forestry/Fruit tree

Livestock/AnimalFishery/Aquaculture

GRAS-Di® Analysis Service - Deliverables⚫ Raw sequence data in FASTQ format⚫ Genotyping data by TOYOTA software with Dominant markers(Optionally)• Genotyping result with Co-dominant markers* Available only in case of diploids, segregated population & homozygous parents, more than 40
samples of F2 generation.

•Mapping result of dominant markers to the reference genome* Available only for the species whose reference genome is available•

Mapping & SNP call result(1) Mapping result (BAM format)(2) SNP call result (VCF format)

Appendix; Result of TOYOTA software⚫ Genotyping through “ Amplicons Presence or absence”.
⚫ GRAS-Di” SNP markers are proviced in csv file.

Intel Core i5-4670 3.40GHz
RAM 8GB

9Appendix; Result of TOYOTA software

10Appendix; Marker detection strategy vs SNPs
⚫ Genotypes of each locus are determined by GRAS-Di software based on thepresence or absence of amplicons (amplicon is considered as locus).
⚫ When amplicon sequences are different between parents, and a reference genome
is available, then the amplicons are available for SNP calling.

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Appendix; Amplicon Mapping on
Chromosome

Multiple candidates of mapped positions for each amplicon are displayed•••ref ID: Chromosome namepos: coordinate position on the chromosome
num mistakes: Number of mismatch bases with reference genomic sequence
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GRAS-Di® Analysis Service
Please Contact to nearest Eurofins Genomics

For APAC Region

For EU Region

Eurofins Genomics
K.K., Japan

Eurofins Genomics
Germany GmbHE-mail:ngs-jp@eurofins.com

E-mail:projectsales-eu@genomics.eurofinseu.com