Prospect and challenge of detecting dynamic gene copy number increases in stem cells by whole genome sequencing

Ulrike Fischer,Andreas Keller,Eckart Meese,Christina Backes,Valentina Galata,Tobias Fehlmann

doi:10.1007/s00109-019-01792-y

Abstract

Gene amplification is an evolutionarily well-conserved and highly efficient mechanism to increase the amount of specific proteins. In humans, gene amplification is a hallmark of cancer and has recently been found during stem cell differentiation. Amplifications in stem cells are restricted to specific tissue areas and time windows, rendering their detection difficult. Here, we report on the performance of deep WGS sequencing (average 82-fold depth of coverage) on the BGISEQ with nanoball technology to detect amplifications in human mesenchymal and neural stem cells. As reference technology, we applied array-based comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), and qPCR. Using different in silico strategies for amplification detection, we analyzed the potential of WGS for amplification detection. Our results provide evidence that WGS accurately identifies changes of the copy number profiles in human stem cell differentiation. However, the identified changes are not in all cases consistent between WGS and aCGH. The results between WGS and the validation by qPCR were concordant in 83.3% of all tested 36 cases. In sum, both genome-wide techniques, aCGH and WGS, have unique advantages and specific challenges, calling for locus-specific confirmation by the low-throughput approaches qPCR or FISH.Key messagesWGS allows for the identification of dynamic copy number changes in human stem cells.Less stringent threshold setting is crucial for detection of copy number increase.Broad knowledge of dynamic copy number is pivotal to estimate stem cell capabilities.

Highlights

Gene amplification is an evolutionarily well-conserved mechanism to allow a highly efficient increase of the amount of specific proteins
whole genome sequencing (WGS) was done by a BGISEQ-500RS on different human stem cells that harbor gene amplifications as shown by independent methods
Stem cells included human mesenchymal stem cells (hMSCs) that were previously analyzed by arraybased comparative genomic hybridization (aCGH), human neural stem cells (hNSCs) that were analyzed using qPCR, and peripheral blood lymphocytes (PB) from a healthy female donor as control

Summary

Introduction

Gene amplification is an evolutionarily well-conserved mechanism to allow a highly efficient increase of the amount of specific proteins. The fact that gene amplification in mammalian stem cells seems to be restricted both regionally, i.e., to specific tissue areas, and temporally, i.e., to specific time windows, complicates the identification of this phenomenon To address this challenge, gene amplification was identified by single cell analysis, for example, by in situ hybridization, as previously shown for CDK4 and MDM2 in mesenchymal stem cells and ERBB2 in trophoblasts [7, 8]. Gene amplification was identified by single cell analysis, for example, by in situ hybridization, as previously shown for CDK4 and MDM2 in mesenchymal stem cells and ERBB2 in trophoblasts [7, 8] These approaches, require a prior knowledge of the amplified locus. These results underline the challenges associated with the use of WGS for the detection of amplifications that are masked by their time and space-limited appearance

Methods

Results

Conclusion