Whether used for the discovery and identification of SNPs or as a screen for panels of thousands of known markers, genotyping by sequencing (GBS) using next-generation sequencing technologies is becoming increasingly important as a cost-effective and unique tool for association studies and genomics-assisted breeding in a range of plant species—including those with complex genomes that lack a reference sequence. The Ion PGM™ Sequencer is the perfect tool for GBS due to its scalability, simplicity, and speed. 

Read the interview with Dr. Jesse Poland (USDA) discussing the advantages of genotyping by sequencing in his work with wheat and barley

What are the advantages of genotyping by sequencing with Ion PGM™ Sequencer?

The Ion Torrent machine
  • Faster, simpler protocol than traditional restriction site associated DNA (RAD) method or full de novo sequencing
  • Allows de novo marker (SNP) discovery, even in cases where there is no reference genome
  • High accuracy of SNP calling
  • Low cost makes it an attractive option for large number of markers or individuals
  • Low amounts of input DNA needed
  • Simplified computational analysis
  • One instrument, many plant applications: de novo and targeted resequencing, transcriptome sequencing, etc. View all

Have a sales rep contact me

Overview of Genotyping by Sequencing Workflows

The workflow below illustrates two different approaches to genotyping by sequencing with the Ion PGM™ System:

  • Restriction Enzyme digestion approach (A): In plants that have no specific SNPs identified, this workflow is ideal for discovering new markers for programs of marker-assisted selection. The complexity of the genome is reduced by digesting the DNA with one or two selected restriction enzymes prior to the ligation of the adapters.
  • Multiplex enrichment PCR (B): In cases where a set of SNPs has been defined for a section of the genome, this approach uses PCR primers designed to amplify the areas of interest.

Genotyping by Sequencing Workflows: Day 1

1 DNA Isolation and Purification

Life Technologies offers an extensive portfolio of kits and reagents for this step of the workflow.

Select the right DNA purification solution here.

2 RE Digestion / Enrichment PCR

A. SNP Discovery: Restriction of gDNA with restriction enzyme, amount and type depend on actual plant species.

B. SNP Validation: Amplification of regions of interest with specific primer sets and incorporate barcodes.

3 Ligate Barcoded Adapters

A. Ligation of sequencing adapters specific to restriction site; adapters contain barcode sequence.

B. Ligation of sequencing adapters (specific to common anchor sequences from step 2).

4 Pre-amplify Fragments

In case of two restriction sites a pre-amplification of the desired restriction fragment is required, (fragment that has both restriction sites).

Day 2

5 Prepare Template

The Ion OneTouch™ System for template preparation is fully automated and requires just minutes of hands-on time

6 Run Sequence

The Ion PGM™ Sequencer is ideal for GBS applications as well as sequencing small genomes, sets of genes, or performing gene expression profiling. See all you can do for plant applications here.

7 Analyze Data

Torrent Suite analysis software delivers significant improvement in base-call accuracy while cutting analysis time to a minimum.

GBS with the Ion PGM™ Sequencer: Case Studies

 


  • Barley (Hordeum vulgare): Drs. Nils Stein (IPK, Gatersleben, Germany) and Jesse Poland (USDA-ARS2, Manhattan, KS, USA) partnered with Life Technologies to develop a protocol for GBS in barley using two restriction enzymes. From their first pilot, they concluded that Ion PGM™ Sequencer has a great potential for large GBS studies due to the high SNP calling accuracy, attractive cost per sample, and unmatched speed in the sequencing workflow.

    Learn more

    Interview with Dr. Poland

 

Ordering Information

Sku Name Size Price Qty
4471269 Ion Xpress™ Plus Fragment Library Kit 10 reactions USD 750.00