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GRAS-Di® for Rapid Genotyping & Genome Wide Marker Discovery

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GRAS-Di® (Genotyping Random Amplicon Sequence-Direct) is an improved method for performing reduced representation sequencing by Next Generation Sequencing. The main applications include marker discovery and genotyping (SNPs) in agricultural or biodiversity studies, in order to identify genetic variation within populations, or develop marker-trait associations for selective breeding purposes.

GRAS-Di has already been successfully used in >60 species including crops, livestock, forestry and aquaculture, and including diploid as well as highly polyploidy species.





  • A simple NGS library preparation method using random primer amplification.
  • Reduces missing data between samples and ensures higher coverage uniformity between samples than standard restriction-digest methods for reduced representation sequencing (RAD-Seq, ddGBS).
  • Rapid and cost-effective.
  • Can be applied to any species with or without a reference genome in natural or segregated populations.




GRAS-Di® amplifies random regions across the genome using random primers. Samples are pooled and the amplicons are sequenced using Illumina Next Generation Sequencing. Sequencing reads are aligned between samples to identify and genotype SNPs.

▽High concentration primers were designed from consensus sequence of adaptors.
sample# Illumina platform Lane Sequence data
96 HiSeq4000 1 500M reads/total
192 HiSeq4000 2 1G reads/total
1000 NovaSeq6000 1 4-5G reads/total




  1. Raw data in FASTQ format
  2. Genotyping result with Dominant markers
  3. Genotyping result with Co-dominant markers
    * Available only in case of diploids, segregated population & homozygous parents, more than 40 samples of F2 generation.
  4. Mapping result of dominant markers to the reference genome
    * Available only for the species whose reference genome is available

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


Case Studies


Sugar Cane variety NiF8 and Ni9 and 22 progenies were analysed with technical replicates (red boxes) by sequencing 32 samples in one lane on HiSeq 2500. The presence (pink) or absence (green) of markers were identified by the achieved read depth (Fig.1).
The method was able to detect 8,683 and 11,655 markers in the parental lines with high reproducibility (N=2, 99.96%). Mapping onto the reference sequence shows a very uniform distribution over the chromosomes(Fig.2). From TOYOTA Presentation at PAG2018 (The Plant and Animal Genomics XXVI Conference).




Q1 How many different amplicon species and how many SNP markers can be obtained by using GRAS-Di?
A1 The number of different amplicon species and SNPs that can be identified by GRAS-Di depends on the genome size of the sample species, the population type (segregated or natural), genetic diversity of the population, and amount of sequence data for each sample. As an example, if you sequence 2M read pairs (4 M reads, 0.4Gb) per sample, 100,000 to 1 million different amplicon species can be obtained, though this can be tuned using different primer pairs, according to the needs of the study. We set a threshold for SNP calling at >30X coverage, which will typically identify approximately 10,000 SNPs, though this depends greatly on the SNP density within the target species. Experimental parameters may be adjusted according to the expected number of SNPs.
Q2 Do you have any recommendation about sample multiplexing?
A2 The current protocol recommendation requires a minimum of 2M read pairs (4M reads, 0.4Gb using 100bp read length) per sample. Illumina HiSeq 4000 can generate an average of 40-50Gb raw data per sequencing lane, so can accommodate 96 samples.
Q3 Are co-dominant markers detectable by GRAS-Di analysis?
A3 Yes, co-dominant markers are detectable in diploids, from segregated population and homozygous parents.
Q4 Can you provide any references citing the use of GRAS-Di technology?
A4 pdf Please refer a Table (PDF).


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