Dataset Information

Chromosomal scan of single sperm cells by combining fluorescence-activated cell sorting and next-generation sequencing.

ABSTRACT:

Purpose

The purpose of this study was to develop a feasible approach for single sperm isolation and chromosome analysis by next-generation sequencing (NGS).

Methods

Single sperm cells were isolated from semen samples of normozoospermic male and an infertile reciprocal translocation (RcT) carrier with the 46,XY,t(7;13)(p12;q12.1) karyotype using the optimized fluorescence-activated cell sorting (FACS) technique. Genome profiling was performed using NGS.

Results

Following whole-genome amplification, NGS, and quality control, the final chromosome analysis was performed on 31 and 6 single cell samples derived from the RcT carrier and normozoospermic male, respectively. All sperm cells from normozoospermic male showed a normal haploid 23-chromosome profile. For the RcT carrier, the sequencing data revealed that 64.5% of sperm cells harbored different variants of chromosome aberrations, involving deletion of 7p or 7q, duplication of 7p, and duplication of 13q, which is concordant with the expected chromosome segregation patterns observed in balanced translocation carriers. In one sample, a duplication of 9q was also detected.

Conclusions

We optimized FACS protocol for simple and efficient isolation of single human sperm cells that subsequently enabled a successful genome-wide chromosome profiling and identification of segmental aneuploidies from these individual cells, following NGS analysis. This approach may be useful for analyzing semen samples of infertile men or chromosomal aberration carriers to facilitate the reproductive risk assessment.

SUBMITTER: Tran QT

PROVIDER: S-EPMC6338602 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Chromosomal scan of single sperm cells by combining fluorescence-activated cell sorting and next-generation sequencing.

Tran Quoc Ty QT Jatsenko Tatjana T Poolamets Olev O Tšuiko Olga O Lubenets Dmitri D Reimand Tiia T Punab Margus M Peters Maire M Salumets Andres A

Journal of assisted reproduction and genetics 20181109 1

<h4>Purpose</h4>The purpose of this study was to develop a feasible approach for single sperm isolation and chromosome analysis by next-generation sequencing (NGS).<h4>Methods</h4>Single sperm cells were isolated from semen samples of normozoospermic male and an infertile reciprocal translocation (RcT) carrier with the 46,XY,t(7;13)(p12;q12.1) karyotype using the optimized fluorescence-activated cell sorting (FACS) technique. Genome profiling was performed using NGS.<h4>Results</h4>Following who ...[more]

PMID: 30411275

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Project description:The elucidation of the cellular processes involved in vitamin and cofactor biosynthesis is a challenging task. The conventional approaches to these investigations rely on the discovery and purification of the products (i.e proteins and metabolites) of a particular transport or biosynthetic pathway, prior to their subsequent analysis. However, the purification of low-abundance proteins or metabolites is a formidable undertaking that presents considerable technical challenges. As a solution, we present an alternative approach to such studies that circumvents the purification step. The proposed approach takes advantage of: (1) the molecular detection capabilities of a riboswitch-based sensor to detect the cellular levels of its cognate molecule, as a means to probe the integrity of the transport and biosynthetic pathways of the target molecule in cells, (2) the high-throughput screening ability of fluorescence-activated cell sorters to isolate cells in which only these specific pathways are disrupted, and (3) the ability of next-generation sequencing to quickly identify the genes of the FACS-sorted populations. This approach was named "RiboFACSeq". Following their identification by RiboFACSeq, the role of these genes in the presumed pathway needs to be verified through appropriate functional assays. To demonstrate the utility of our approach, an adenosylcobalamin (AdoCbl)-responsive riboswitch-based sensor was used in this study to demonstrate that RiboFACSeq can be used to track and sort cells carrying genetic mutations in known AdoCbl transport and biosynthesis genes with desirable sensitivity and specificity. This method could potentially be used to elucidate any pathway of interest, as long as a suitable riboswitch-based sensor can be created. We believe that RiboFACSeq would be especially useful for the elucidation of biological pathways in which the proteins and/or their metabolites are present at very low physiological concentrations in cells, as is the case with vitamin and cofactor biosynthesis.

Dataset Information

Chromosomal scan of single sperm cells by combining fluorescence-activated cell sorting and next-generation sequencing.

Purpose

Methods

Results

Conclusions

Publications

Chromosomal scan of single sperm cells by combining fluorescence-activated cell sorting and next-generation sequencing.

Similar Datasets

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