A new mouse SNP genotyping assay for speed congenics: combining flexibility, affordability, and power

Kimberly R Andrews,Matthew W Fagnan,Sarah M Garrison,Shirley Luckhart,Delaney Wagers,Daniel D New,Gretchen Hansten,Brandi K Torrevillas,Nora Céspedes,Jessica Strickland,Samuel S Hunter

doi:10.1186/s12864-021-07698-9

Abstract

BackgroundSpeed congenics is an important tool for creating congenic mice to investigate gene functions, but current SNP genotyping methods for speed congenics are expensive. These methods usually rely on chip or array technologies, and a different assay must be developed for each backcross strain combination. “Next generation” high throughput DNA sequencing technologies have the potential to decrease cost and increase flexibility and power of speed congenics, but thus far have not been utilized for this purpose.ResultsWe took advantage of the power of high throughput sequencing technologies to develop a cost-effective, high-density SNP genotyping assay that can be used across many combinations of backcross strains. The assay surveys 1640 genome-wide SNPs known to be polymorphic across > 100 mouse strains, with an expected average of 549 ± 136 SD diagnostic SNPs between each pair of strains. We demonstrated that the assay has a high density of diagnostic SNPs for backcrossing the BALB/c strain into the C57BL/6J strain (807–819 SNPs), and a sufficient density of diagnostic SNPs for backcrossing the closely related substrains C57BL/6N and C57BL/6J (123–139 SNPs). Furthermore, the assay can easily be modified to include additional diagnostic SNPs for backcrossing other closely related substrains. We also developed a bioinformatic pipeline for SNP genotyping and calculating the percentage of alleles that match the backcross recipient strain for each sample; this information can be used to guide the selection of individuals for the next backcross, and to assess whether individuals have become congenic. We demonstrated the effectiveness of the assay and bioinformatic pipeline with a backcross experiment of BALB/c-IL4/IL13 into C57BL/6J; after six generations of backcrosses, offspring were up to 99.8% congenic.ConclusionsThe SNP genotyping assay and bioinformatic pipeline developed here present a valuable tool for increasing the power and decreasing the cost of many studies that depend on speed congenics. The assay is highly flexible and can be used for combinations of strains that are commonly used for speed congenics. The assay could also be used for other techniques including QTL mapping, standard F2 crosses, ancestry analysis, and forensics.

Highlights

Speed congenics is an important tool for creating congenic mice to investigate gene functions, but current Single nucleotide polymorphism (SNP) genotyping methods for speed congenics are expensive
Bioinformatic analysis: genotyping We developed a bioinformatic pipeline that analyzes the sequence data generated by our assay, producing output that can be interpreted to aid in practical decisionmaking for speed congenics experiments (Fig. 1)
The majority of SNPs had more than ten mapped sequence reads for all samples, except one poor-performing sample in the first batch for which most SNPs had fewer than ten reads (Fig. 2)

Summary

Introduction

Speed congenics is an important tool for creating congenic mice to investigate gene functions, but current SNP genotyping methods for speed congenics are expensive. These methods usually rely on chip or array technologies, and a different assay must be developed for each backcross strain combination. Speed congenics uses genetic markers to identify backcross offspring with the highest levels of ancestry for the desired genetic background. By preferentially selecting these individuals for the backcross step, the number of generations required to develop congenic mice can be reduced from ten to five

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