SSRgenotyper: A new tool to digitally genotype simple sequence repeats

February 05, 2021

SSRgenotyper is a newly developed, free bioinformatic tool that allows researchers to digitally genotype sequenced populations using simple sequence repeats (SSRs), a task that previously required time-consuming lab-based methods.

Reporting in a recent issue of Applications in Plant Sciences, the tool's developers designed the program to seamlessly integrate with other applications currently used for the detection and analysis of SSRs.

Simple sequence repeats are short chains of repeating nucleotides that are prone to mutation. The variability of these DNA sequences makes them ideal for genetic analyses to distinguish between individuals and are often the marker of choice for paternity and forensic testing.

In research fields, SSRS have the added benefit of being selectively neutral, meaning they don't code for any physical traits and therefore aren't subject to most types of natural selection, making them an excellent tool to study populations without the obscuring effects of convergent evolution.

Recent advances in next-generation sequencing have helped streamline the process of SSR identification, especially in model organisms or groups with an available reference genome assembly. As technology continues to improve and sequencing costs decrease, sequencing large portions of a genome for the purposes of SSR analysis, even in non-model organisms, is becoming more feasible and widespread in the scientific literature.

However, the process of genotyping -- determining which individuals have which alleles -- still relies predominantly on visualizing amplified DNA on an electrophoresis gel, an involved and potentially hazardous process, as DNA fragments are often stained with carcinogenic chemicals.

It also has the added issue of alleles being measured based on the size of the resulting bands, which is an estimate for the number of nucleotides in the amplified DNA fragment. Because there may be slight variations in the flanking regions that surround the SSRs of interest, and because there is no standardized method of determining an allele's size using these methods, genotyping results from one experiment cannot be easily transferred or compared to those of another experiment.

The development of SSRgenotyper renders such lab-based efforts obsolete. By working in tandem with other bioinformatic programs that detect SSRs in reference DNA and programs that align sequence data from target populations with the corresponding SSR reference file, SSRgenotyper is able to quickly genotype all SSRs for each individually sequenced sample.

"SSRgenotyper goes the next step by genotyping SSRs within sequenced populations -- strictly from sequencing data (no PCR or electrophoresis)," said Jeff Maughan, a professor of Plant and Wildlife Sciences at Brigham Young University and senior author of the study. "The output from SSRgenotyper are files ready for population genetic analysis or linkage map formation."

Not only does the program reduce the amount of time and work required to genotype populations, it also solves the transferability problem inherent in electrophoresis estimates by directly counting the total number of base pairs in a given sequence repeat.

"Since the SSRs are genotyped based on the number of repeated motifs at the SSR locus and not on the PCR product size, the allele calls are standardized and transferable from project to project or from lab to lab," said Maughan.

The program, which is coded in Python 3, requires only three positional arguments to run, provides the option to specify several conditional arguments (such as percentage thresholds for heterozygosity, the size of the flanking regions, and for the removal of spurious alleles), and can be performed on a regular desktop computer.

Once complete, SSRgenotyper generates multiple file types, including basic summary and statistical files, as well as a .pop, a .map, and an alignment file formatted for use in additional programs to facilitate downstream analyses.

As a proof of concept, Maughan and his colleagues tested SSRgenotyper's accuracy at correctly determining an individual's genotype by running the program on publicly available sequences of quinoa (Chenopodium quinoa) and the oat species Avena atlantica. The resulting accuracy rate was 97% or greater, which increased with the inclusion of additional sequence reads.

With the continued development and efficiency of next-generation sequencing methods, tools like SSRgenotyper seem poised to reduce the amount of lab work required in genetic studies.

"Sequencing is already the method of choice in most genetic research projects," said Maughan. "As costs continue to drop and new bioinformatic tools are developed, it is highly likely that future population genetics studies will be based solely on next-generation sequencing -- completely avoiding the cumbersome tasks of PCR and electrophoresis."
-end-
Citation: Lewis, D. H., D. E. Jarvis, and P. J. Maughan. 2020. SSRgenotyper: A simple sequence repeat genotyping application for whole-genome resequencing and reduced representational sequencing projects. Applications in Plant Sciences 8(12): e11402. https://doi.org/10.1002/aps3.11402

Applications in Plant Sciences (APPS) is a monthly, peer-reviewed, open access journal focusing on new tools, technologies, and protocols in all areas of the plant sciences. It is published by the Botanical Society of America (http://www.botany.org), a nonprofit membership society with a mission to promote botany, the field of basic science dealing with the study and inquiry into the form, function, development, diversity, reproduction, evolution, and uses of plants and their interactions within the biosphere. APPS is available as part of the Wiley Online Library (https://onlinelibrary.wiley.com/journal/21680450).

For further information, please contact the APPS staff at apps@botany.org.

Botanical Society of America

Related Genotype Articles from Brightsurf:

Genetics: Romantic relationship dynamics may be in our genes
Variations in a gene called CD38, which is involved in attachment behaviour in non-human animals, may be associated with human romantic relationship dynamics in daily life, according to a study published in Scientific Reports.

Alport syndrome: Research highlights link between genotype and treatment effectiveness
A large-scale analysis of the clinical characteristics of Alport syndrome in Japanese patients has revealed for the first time in the world that the effectiveness of existing treatment with ACE inhibitors and/or angiotensin receptor blockers (RAS inhibitors) varies depending on the type of mutation in the syndrome's causal gene (COL4A5).

Function-based sequencing technique permits analysis of just a single bacteria cell
A new function-based sequencing technique using optical tweezers and taking advantage of the properties of gravity is letting researchers analyze bacteria cells one by one.

Alport syndrome severity can be predicted by causative protein genotype
Researchers from Kumamoto and Kobe Universities in Japan have successfully developed a system for predicting the severity of Alport syndrome, a serious hereditary kidney disease.

Evolutionary changes in brain potentially make us more prone to anxiety
Neurochemicals such as serotonin and dopamine play crucial roles in cognitive and emotional functions of our brain.

Genetics of nephropathia epidemica researched by Kazan Federal University
This particular inquiry pertains to CCR5 -- a gene which has lately been on the focuses of research worldwide.

New test offers improved diagnosis and management of chronic hepatitis B
A report in The Journal of Molecular Diagnostics, published by Elsevier, describes a new and powerful laboratory tool that may improve the diagnosis and treatment of hepatitis B virus (HBV) infection.

Happy in marriage? Genetics may play a role
People fall in love for many reasons -- similar interests, physical attraction, and shared values among them.

Enlarged genotype-phenotype correlation for a deletion in neurofibromatosis type 1
International collaborative research led by Ludwine Messiaen, Ph.D., shows that while a three-base pair, in-frame deletion called p.Met992del in the NF1 gene has a mild phenotype for people with the genetic disorder neurofibromatosis type 1, or NF1, the mutation does cause complications.

Novel information about the effects of in vitro fertilization on embryonic growth
In vitro fertilization affects the regulatory region of genes essential for placental and embryonic growth, as well as the birth weight.

Read More: Genotype News and Genotype Current Events
Brightsurf.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.