Insights from Initial Variant Detection by Sequencing Single Sperm in Cattle

Liu Yang,Yahui Gao,George E Liu,Li Ma,Hongping Zhang,Clarissa Boschiero,Li Li

doi:10.3390/dairy2040050

Abstract

Meiotic de novo mutation (DNM) is one of the important phenomena contributing to gamete genome diversity. However, except for humans and a few model organisms, they are not well studied in livestock, including cattle. Moreover, bulk sperm samples have been routinely utilized in experiments, which include millions of single sperm cells and only report high-frequency variants. In this study, we isolated and sequenced 143 single sperms from two Holstein bulls and identified hundreds of candidate DNM events in ten sperms with deep sequencing coverage. We estimated DNM rates ranging from 1.08 × 10−8 to 3.78 × 10−8 per nucleotide per generation. We further validated 12 out of 14 selected DNM events using Sanger sequencing. To our knowledge, this is the first single sperm whole-genome sequencing effort in livestock, which provided useful information for future studies of point mutations and male fertility. Our preliminary results pointed out future research directions and highlighted the importance of uniform whole genome amplification, deep sequence coverage, and dedicated software pipelines for genetic variant detection using single-cell sequencing data.

Highlights

Recent breakthroughs in the development and application of single-cell sequencing technologies provide an avenue for dissecting population lineages and heterogeneity and understanding cell identity, differentiation, and function [1,2,3,4,5]
Single-cell DNA-seq technologies produce data, which allow the detection of single nucleotide variants (SNVs) and short insertion and deletion (INDEL) variants, together with structural variations or abnormal chromosome numbers on the single-cell level [6,7,8]
We identified hundreds of candidate de novo mutation (DNM) events in ten sperms with deep sequencing coverage by comparing them to the somatic genome

Summary

Introduction

Recent breakthroughs in the development and application of single-cell sequencing technologies provide an avenue for dissecting population lineages and heterogeneity and understanding cell identity, differentiation, and function [1,2,3,4,5]. Sperm is one of the most important cells because it delivers the entire paternal genetic materials to the offspring. As novel mutations can occur during gametogenesis and postzygotically, studying mutations in sperm is important for male fertility. Recent genome and exome sequencing studies of parent–offspring trios have provided the first insights into the number and distribution of the de novo mutations (DNMs) [9]. DNMs have been shown to be a major cause of severe early-onset genetic disorders such as intellectual

Methods

Results

Conclusion