High-throughput whole-genome sequencing of E14 mouse embryonic stem cells

Abstract

Mouse E14 embryonic stem cells (ESCs) are the most used ESC line, often employed for genome-wide studies involving next generation sequencing analysis [1], [2], [3], [4], [5]. More than 2 × 10 E9 sequences made on Illumina platform derived from the genome of E14 embryonic stem cells cultured in our laboratory were used to build a database of about 2.7 × 10 E6 single nucleotide variant [6]. The database was validated using other two sequencing datasets from other laboratory and high overlap was observed. The identified variants are enriched on intergenic regions, but several thousands reside on gene exons and regulatory regions, such as promoters, enhancers, splicing site and untranslated regions of RNA, thus indicating high probability of an important functional impact on the molecular biology of these cells. We created a new E14 genome assembly including the new identified variants and used it to map reads from next generation sequencing data generated in our laboratory or in others on E14 cell line. We observed an increase in the number of mapped reads of about 5%. CpG dinucleotide showed the higher variation frequency, probably because it could be a target of DNA methylation. Data were deposited in GEO datasets under reference GSM1283021 and here: http://epigenetics.hugef-research.org/data.php.

Specifications
Organism/cell line/tissue	Mouse E14 embryonic stem cells
Sex	Male
Sequencer or array type	Illumina HiScanSQ
Data format	Raw and analyzed
Experimental factors	N/A
Experimental features	Whole genome sequencing of E14 embryonic stem cells
Consent	N/A
Sample source location	Torino, Italy

Direct link to deposited data

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1283021

http://epigenetics.hugef-research.org/data.php

Experimental design, materials and methods

E14 mouse ES cells were cultured in ESC medium (DMEM high glucose with 15% fetal bovine serum [FBS], NNEA1x, NaPyr1x, 0.1 mM 2-mercaptoethanol, and 1500 U/ml LIF). Genomic DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen).

For sequencing of E14 genome, DNA was sonicated for 17′ pulse 30″ON/30″OFF high with Bioruptor Twin (Diagenode). Libraries were generated with DNA Sample Prep Kit (Illumina) and sequenced on Illumina HiScanSQ Platform. Basecalls performed using CASAVA version 1.8 following default parameters. Reads quality was estimated using FastQC tool v0.10.1 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Nucleotide positions with a quality score under 30 (Phred33 scale) were trimmed using the fastx_trimmer tool from the FASTX Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).

After low-quality positions trimming, reads in which sequencing continued through the 3′ adapter sequence were clipped using the fastx_clipper tool from the FASTX Toolkit. Then, reads were aligned to the mouse genome assembly mm9 using Bowtie [7] v0.12.7 with the following parameters: -q --max /dev/null -v 1 -S --sam-nohead -m 1. Reads with the same mapping positions were collapsed into one using the rmdup tool from SAMtools. Variants calling was performed using the mpileup tool from SAMtools [8]. Next, we used VCFtools [9] v0.1.11 (http://vcftools.sourceforge.net/) to select only SNVs with coverage of ≥10 and a frequency of ≥0.5. Moreover, using custom Perl scripts we discarded sites with more than one variant call at the same place. Finally, using the GATK v2.7-4 (http://www.broadinstitute.org/gatk/) FastaAlternateReferenceMaker function we created the new reference E14 assembly from the mm9 genome assembly.

These data can be found at: http://epigenetics.hugef-research.org/data.php.