Sequence-based genotyping methods

For some applications, NGS offers a lower cost than arrays for studying genetic variation

Genotyping by Sequencing

Historically, array-based approaches to single nucleotide polymorphism (SNP) screening have been the method of choice in analyzing and associating traits with regions of the genome for many plants and animals. As sequencing costs continue to decline, researchers are developing new approaches that leverage next-generation sequencing (NGS) for genotyping.

Genotyping by sequencing, or next-generation genotyping, is a genetic screening method for discovering novel plant and animal SNPs and performing genotyping studies. For some applications, such as genotype screening and genetic mapping, sequence-based genotyping provides a lower-cost alternative to arrays for studying genetic variation.

Genotyping by Sequencing in 3 Simple Steps

Sequence predetermined areas of genetic variation over many samples with this seamless workflow solution.

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  • Sequences predetermined areas of genetic variation over many samples
  • Provides a low cost per sample for certain applications
  • Reduces ascertainment bias compared to arrays
  • Identifies variants other than SNPs, including small insertions, deletions, and microsatellites
  • Enables comparative analyses across samples in the absence of a reference genome
  • Informs genetic mapping, screening backcross lines, purity testing, constructing haplotype maps, association mapping, and genomic selection for plant studies
Advantages of Genotyping by Sequencing

Genotyping by sequencing is cost-effective for populations with complex genomes or limited available resources. Techniques include amplicon-based targeted sequencing, hybridization-based enrichment sequencing, and restriction enzyme reduced representation sequence-based genotyping.

For optimal results using this method, Illumina recommends having:
  • A reference genome
  • High-diversity samples
  • Finely tuned coverage across multiplexed samples
  • Variant call format (VCF) file converter
  • Tolerance of ambiguity in heterozygote detection and redundancy checks to minimize false positives

Various sequence-based genotyping approaches have been developed.1–4 Enrichment methods are useful for plants, which often contain duplicated areas of the genome. Restriction enzyme methods are advantageous for species where there is no prior knowledge of the genome.

Genotyping by Sequencing Methods

Illumina sequencing by synthesis (SBS) chemistry is the most widely adopted NGS technology, generating approximately 90% of global sequencing data.*

In addition to our industry-leading data quality, Illumina offers integrated sequencing workflows that simplify the entire process, from library preparation to data analysis and biological interpretation.

Click on the below to view products for each workflow step.

Design Studio

An easy-to-use online software tool that provides dynamic feedback to optimize probe designs.

TruSeq Custom Amplicon Library Prep Kit

Fully customizable, amplicon-based assay for targeted resequencing.

Nextera XT DNA Library Prep Kit

Library preparation for small genomes (bacteria, archaea, viruses), amplicons, and plasmids in less than 90 minutes.

Library Prep Kit Selector Library Prep Kit Selector

Determine the best kit for your needs based on project type, starting material, and method or application.

MiSeq Series

Speed and simplicity for targeted and small genome sequencing.

NextSeq Series

Flexible power for sequencing a whole genome or up to 384 amplicons per run.

HiSeq Series
Power and efficiency for large-scale genomics.
NovaSeq Series

Scalable throughput and flexibility for virtually any genome, sequencing method, and scale of project.

Platform Comparison Tool

Compare sequencing platforms and identify the best system for your lab and applications.

Sequencing Reagents

Find kits that include sequencing reagents, flow cells, and/or buffers tailored to each Illumina sequencing system.

TruSeq Amplicon App

Streamlined analysis of NGS data enriched for particular target sequences using TruSeq Amplicon reads.

BWA Whole-Genome Sequencing App

Processes whole-genome sequencing data using BWA alignment and GATK variant calling.

Isaac Whole-Genome Sequencing App

Quickly extracts biological information from whole-genome sequences, using Isaac alignment and variant calling.

BaseSpace Sequence Hub

The Illumina genomics computing environment for NGS data analysis and management.

BaseSpace Correlation Engine

A growing library of curated genomic data to support researchers in identifying disease mechanisms, drug targets, and biomarkers.

For small genomes (eg, Drosophila) or high-profile research species (eg, Arabidopsis), genotyping and variant screening can be completed using standard whole-genome sequencing or resequencing methods relative to a reference. Low-depth sequencing can be used for phylogenetic or comparative analyses.

Learn More
Genotyping by Sequencing for Small Genomes
Plant and Animal Sequencing

NGS technology can benefit agricultural studies of plants and animals, whether it is used for de novo sequencing, transcriptome analysis, genotyping by sequencing, or metagenomics. Learn more about plant and animal sequencing.

NGS technology can benefit agricultural studies
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Genotyping by Sequencing in Agrigenomics
Genotyping by Sequencing in Agrigenomics

This application spotlight describes the advantages and considerations of several sequence-based genotyping research methods.

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Revitalizing Fish Populations With NGS
Revitalizing Fish Populations With NGS

Scientists developed Genotyping-in-Thousands by sequencing (GT-Seq) to genotype thousands of fish simultaneously.

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Amplicon Sequencing
Amplicon Sequencing

Amplicon sequencing is a highly targeted approach that researchers can use to genotype large genomes.

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References
  1. Mamanova L, Coffey AJ, Scott CE, et al. Target-enrichment strategies for next-generation sequencing. Nat Methods. 2010;7:111-118.
  2. Liu S, Yeh CT, Tang HM, Nettleton D, Schnable PS. Gene mapping via bulked segregant RNA-Seq (BSR-Seq). PLoS One. 2012;7:e36406.
  3. Cronn R, Knaus BJ, Liston A, et al. Targeted enrichment strategies for next generation plant biology. Am J Bot. 2012;99:291-311.
  4. Andolfatto P, Davison D, Erezyilmaz D, et al. Multiplexed shotgun genotyping for rapid and efficient genetic mapping. Genome Res. 2011;21:610-617.

*Data calculations on file. Illumina, Inc., 2015