Genotyping By Sequencing (GBS)

• As an ultimate Marker Assisted Selection tool and a cost-effective technique, GBS has been successfully used in implementing genome-wide association study (GWAS), genomic diversity study, genetic linkage analysis, molecular marker discovery and genomic selection under a large scale of plant breeding programs. Identification of high density SNP markers through GBS to construct genetic linage maps has a 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. Combination of enzymes set can be used to prepare libraries for parental as well as progeny lines and sequencing on illumina NextSeq 500 or HiSeq 2500 platform using single end library can be carried out by multiplexing for 48, 96, 192 samples or as per request. 

Methodology

Sample Requirement:

• Submit 3-5 µg of RNA-free genomic DNA (NGS grade) of molecular weight >40kb, Nanodrop A_260/280 ratio >1.8; A_260/230=2.0-2.2; and at a concentration of ~50 ng/µl
• Or 5-10 g of fresh tissue fast frozen in liquid nitrogen and shipped in dry ice to Eurofins facility.

Sample QC:

• 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 checking will be carried out using agarose electrophoresis and DIN value estimation using Agilent Tape Station. Quantification will be carried out using 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 will be carried out using ligation of common and barcoded adapters to the RE digested DNA sticky ends and final amplification will be carried out using primers complementary to illumina sequencing flow cell primers.
• Library validation will be carried out using High Sensitivity D1000 Screen tape using Agilent Tape station kit.

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 in link or Pen Drive or Hard Disk.
• For analysis please visit Bioinformatics Services

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 TAT is offered expecting no biological or technical difficulties for processing of all project samples.