Optimizing Cost-Effective gene expression phenotyping approaches in cattle using 3' mRNA sequencing.

Area of Science:

• Livestock Genomics
• Molecular Biology
• Bioinformatics

Background:

• Genetic and genomic selection in livestock requires extensive phenotyping, which is often costly and difficult for traits like meat quality or disease resistance.
• Leveraging molecular intermediates through cost-effective sequencing offers a scalable alternative for capturing crucial animal data.

Purpose of the Study:

• To optimize 3' mRNA sequencing (3' mRNA-Seq) for cost-effective molecular phenotyping in cattle using whole blood samples.
• To identify the most effective library preparation kit and optimal sequencing depth for capturing gene expression phenotypes.

Main Methods:

• Compared Takara SMART-Seq v4 3' DE and Lexogen QuantSeq library preparation kits for 3' mRNA-Seq.
• Performed serial downsampling analysis on sequencing data (0.5 to 20 million reads/sample) from 15 Holstein calves.
• Evaluated metrics including read quality, gene expression, and variant calling.

Main Results:

• Takara SMART-Seq v4 3' DE significantly outperformed Lexogen QuantSeq across all evaluated metrics.
• As few as 8.0 million reads per sample captured substantial gene expression variation, with marginal gains beyond this depth.
• 3' mRNA-Seq data enabled the discovery of numerous SNPs and INDELs suitable for genotype imputation.

Conclusions:

• 3' mRNA-Seq with the Takara SMART-Seq v4 3' DE kit is a highly cost-effective method (<$25/sample) for quantifying molecular phenotypes and enabling genotype imputation in cattle.
• This approach facilitates large-scale molecular phenotyping and genetic improvement in livestock populations.