Genotyping by Sequencing in Plants and Animals

The use of next-generation sequencing technologies for genotyping by sequencing (GBS) is becoming an increasingly important, cost-effective, and unique tool for association studies and genomics-assisted breeding in a range of plant and animal species, including those with complex genomes that lack a reference sequence. It can be used for the discovery and identification of SNPs or to screen large panels of known markers.

The Ion Proton™ and Ion PGM™ Sequencers enable plant and animal researchers to use flexible, high-throughput GBS to perform fast and cost-effective studies.

GBS: advantages of the Ion Proton™ and Ion PGM™ Sequencers

  • Allow both SNP marker confirmation and de novo marker (SNP) discovery, even in cases where there is no reference genome
  • Provide attractive, low-cost options for large numbers of samples and data points 
  • Utilize highly multiplexed sequencing to scale from thousands to millions of data points per day
  • Enable 2–4 hour sequencing turnaround times to facilitate efficient planning for the next reproduction cycle
  • Deliver a high degree of accuracy of SNP calling
  • Offer versatility for many applications: de novo and targeted resequencing, transcriptome sequencing, and more ›

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Two strategies for genotyping by sequencing

The Ion PGM™ and Ion Proton™ Systems support two different GBS strategies:

  • Multiplex enrichment PCR
    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 targets of interest. View our complete solution for multiplex PCR below. Creating and ordering custom panels is easy with the Ion AmpliSeq™ Designer—a free, online assay design tool that enables custom design for any genome. Use the preloaded bovine, chicken, pig, sheep, maize, tomato, soybean and rice genomes to design your custom panel, or easily upload a FASTA file with your desired reference sequences for your genome of interest.

    Design your Ion AmpliSeq™ panel here >
  • Improving Plant Breeding Using Ion AmpliSeqTM and Ion Torrent™ Sequencing Technologies for Targeted Re-sequencing and SNP Analysis in Tomato

    Case Study: Improving plant breeding using Ion AmpliSeq™ and Ion Torrent™ sequencing technologies for targeted resequencing and SNP analysis in tomato

    High-throughput characterization of known and novel SNPs is critical for plant genotyping applications. Recent advances in NGS have simplified plant SNP discovery and validation and have also created a demand for high-throughput SNP biomarker screening for plant breeding program improvements. In this study we used the Ion AmpliSeq™ panel workflow to genotype 370 SNPs from 192 matched paired tomato leaf samples (purified DNA and crude leaf lysate) on the Ion PGM™ Sequencer. The results showed a call rate of 94% and a call concordance of 99% between purified DNA and leaf lysates.

    Learn more about this study in our recorded webinar

  • Restriction enzyme (RE) digestion

    In this approach, the complexity of the genome is reduced by digesting the DNA with one or two selected restriction enzymes prior to the ligation of sequencing adapters. Compared to the multiplex PCR approach, which is used for targeting known variants, this method is better suited for situations where little genome sequence is available or no prior SNP variants have been identified, for discovering new markers for marker-assisted selection programs.

  • Wheat and barley genotyping by sequencing

    Case study: Wheat and barley genotyping by sequencing

    Drs. Nils Stein (IPK, Gatersleben, Germany) and Jesse Poland (USDA-ARS2, Manhattan, Kansas, USA) partnered with Thermo Fisher Scientific (formerly Life Technologies) to develop a protocol for GBS in barley using two restriction enzymes. In their first pilot study, they concluded that the Ion PGM™ Sequencer shows great potential for large GBS studies due to the high SNP calling accuracy, attractive cost per sample, and unmatched speed in the sequencing workflow. 

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

    Genotyping by sequencing workflows

    Day 1

    1 DNA preparation

    We offer an extensive portfolio of DNA isolation kits.

    2 RE digestion / multiplex PCR

    A. SNP verification: amplification of targeted regions with custom designed primer sets and incorporated barcodes

    B. SNP discovery: gDNA restriction enzyme digestion

    3 Ligate barcoded adapters

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

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

    4 Amplify library fragments

    For the multiplex PCR approach this step is optional.

    In the RE digestion method, the selected library fragments are amplified using primer sequences common to the restriction sites.

    Day 2

    5 Prepare template

    The Ion Chef™ System for template preparation is fully automated

    6 Sequence

    The Ion Proton™ Sequencer is ideal for GBS applications

    7 Analyze data

    Torrent Suite™ analysis software is used for SNP calls

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