Combined ultra-low input mRNA and whole-genome sequencing of human embryonic stem cells

BMC Genomics

Research Article

Björn Lichtner¹  Mirjam Blattner¹  Hans Lehrach¹  James Adjaye²  Florian Mertes³  Jörg Otte⁴  Wasco Wruck⁴  Bernd Timmermann⁵  Heiner Kuhl⁵ 

[1] Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Ihnestr. 63-73, 14195, Berlin, Germany;Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Ihnestr. 63-73, 14195, Berlin, Germany;Institute for stem cell research and regenerative medicine, Medical Faculty, Heinrich Heine University, Moorenstr. 5, 40225, Düsseldorf, Germany;Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Ihnestr. 63-73, 14195, Berlin, Germany;Molecular Exposomics, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany;Institute for stem cell research and regenerative medicine, Medical Faculty, Heinrich Heine University, Moorenstr. 5, 40225, Düsseldorf, Germany;Next Generation Sequencing Group, Max Planck Institute for Molecular Genetics, Ihnestr. 63-73, 14195, Berlin, Germany;

关键词: Next generation sequencing;    RNA-seq;    Whole-genome sequencing;    Ultra-low input sequencing;    Single cell;    Pluripotency;    Embryonic stem cells;   

DOI  :  10.1186/s12864-015-2025-z

 received in 2015-07-07, accepted in 2015-10-07,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundNext Generation Sequencing has proven to be an exceptionally powerful tool in the field of genomics and transcriptomics. With recent development it is nowadays possible to analyze ultra-low input sample material down to single cells. Nevertheless, investigating such sample material often limits the analysis to either the genome or transcriptome. We describe here a combined analysis of both types of nucleic acids from the same sample material.MethodsThe method described enables the combined preparation of amplified cDNA as well as amplified whole-genome DNA from an ultra-low input sample material derived from a sub-colony of in-vitro cultivated human embryonic stem cells. cDNA is prepared by the application of oligo-dT coupled magnetic beads for mRNA capture, first strand synthesis and 3’-tailing followed by PCR. Whole-genome amplified DNA is prepared by Phi29 mediated amplification. Illumina sequencing is applied to short fragment libraries prepared from the amplified samples.ResultsWe developed a protocol which enables the combined analysis of the genome as well as the transcriptome by Next Generation Sequencing from ultra-low input samples. The protocol was evaluated by sequencing sub-colony structures from human embryonic stem cells containing 150 to 200 cells. The method can be adapted to any available sequencing system.ConclusionsTo our knowledge, this is the first report where sub-colonies of human embryonic stem cells have been analyzed both at the genomic as well as transcriptome level. The method of this proof of concept study may find useful practical applications for cases where only a limited number of cells are available, e.g. for tissues samples from biopsies, tumor spheres, circulating tumor cells and cells from early embryonic development. The results we present demonstrate that a combined analysis of genomic DNA and messenger RNA from ultra-low input samples is feasible and can readily be applied to other cellular systems with limited material available.

【 授权许可】

CC BY   
© Mertes et al. 2015