As an ultimate marker assisted selection tool and a cost-effective technique, Genotyping-by-sequencing (GBS) has been successfully used in implementing genome-wide association studies (GWAS), genomic diversity studies, genetic linkage analysis, molecular marker discovery, and genomic selections for large scale plant breeding programmes. Identifying high-density SNP markers through GBS to construct genetic linage maps has great value for numerous applications in plant breeding.

The method involves cutting down a genome anywhere from 0.1 to 15% with at least one restriction enzyme and sequencing the ends of the resulting fragments for either genetic marker discovery or genotyping. The complexity of the genome in this approach is reduced by digesting the DNA with selected restriction enzymes. The enzyme set can be used to prepare libraries for parental and progeny lines. Sequencing can then be performed on the Illumina NextSeq 500 or HiSeq 2500 platform using single-end libraries, with multiplexing options for 48, 96, or 192 samples, or customised configurations upon request.

Methodology

Sample requirement

• Submit 3-5 µg of RNA-free genomic DNA (gDNA) suitable for next generation sequencing (NGS) with molecular weight >40kb, Nanodrop A260/280 ratio >1.8; A260/230=2.0-2.2; and at a concentration of ~50 ng/µl.
• Alternatively, 5-10 g of fresh tissue fast frozen in liquid nitrogen and shipped in dry ice to our facility.

Sample quality control

• RNA free NGS grade gDNA isolation will be carried out using commercially available kit with further purification making it suitable for NGS library preparation.
• Further quality assessment is performed using agarose gel electrophoresis and DNA Integrity Number (DIN) determination with the Agilent TapeStation system.
• DNA quantification is performed using the Qubit 3.0 Fluorometer.

Library preparation

• Restriction enzyme selection will be carried out to reduce genome complexity and avoid the repetitive fraction of the genome.
• GBS library preparation is performed by ligating common and barcoded adapters to the sticky ends of restriction enzyme (RE)-digested DNA, followed by final amplification using primers complementary to the Illumina sequencing flow cell primers.
• Library validation is performed using the high sensitivity D1000 ScreenTape assay on the Agilent TapeStation system.

Sequencing

• Prepared library will be sequenced using 1 x 75 bp or 1 x 150 single end sequencing using latest sequencing chemistry on NextSeq 500.

Deliverable

• Raw data will be available for download as a compressed archive of FASTQ files for each sample.
• Comprehensive compiled report and data set will be shared through secure file-sharing platform.

Turnaround time

5-7 weeks after arrival of your samples and all necessary information. It depends upon data size, scope, technology selected, number of samples and complexity of the project. The given turnaound time presumes no biological or technical difficulties in the processing of all project samples.