Bioscope software

Genomic DNA extraction from collected tissues was carried out using the ChargeSwitch® gDNA Mini Tissue Kit (Life Technologies, Carlsbad, CA). The extracted DNA was used for construction of a library using a SOLiD Fragment Library Construction Kit (Life Technologies). The library DNA was subjected to whole-exome enrichment using a Sureselect Human All Exon Kit (Agilent Technologies, Inc., Santa Clara, CA). The enriched library DNA was then sequenced using the SOLiD System, a deep sequencer employing the massively parallel sequencing method, and analyzed using Bioscope software (Life Technologies). After excluding mutations such as CSF1R and EIF2B, they were finally identified as AARS2 compound heterozygous mutations. Family members' gene mutations were verified by Sanger sequencing.
For validation of the WES data, DNA was amplified by polymerase chain reaction (PCR). The amplified products were treated with ExoSAP-IT (GE Healthcare, Chalfont St Giles, Buckinghamshire, UK) and sequenced using Bigdye Terminator and a 3130xl Genetic Analyzer (Life Technologies) for examination of mutations in AARS2.

The reads obtained from sequencing were mapped to the zebrafish genome (Danio rerio zv8) using the BioScope software (Life Technologies, Darmstadt, Germany). In addition to the genomic sequence, the reference taken for mapping contained ribosomal and transfer RNA sequences from zebrafish, as well as sequences of the SOLiD adaptors and homopolymer sequences (poly-A, poly-T) were also used in order to filter reads matching these sequences. For the identification of reads covering exon-exon (inter-exon) junctions, sequences were added to the reference representing all possible combinations of two exons from each gene based on the zebrafish annotation (zv8; UCSC Genome Browser).

Nuclear extracts from four E12.5 embryonic hearts were used in each native ChIP experiment following a previously described protocol with minor modifications (Nimura et al., 2006 (link); 2009 (link)). Isolated nuclei from embryonic hearts were treated at 25°C for 30 min with 4.8 U ml⁻¹ micrococcal nuclease in 250 µl of a nuclear isolation buffer containing 400 mM NaCl, which was then diluted to 200 mM NaCl. The digested chromatin was immunoprecipitated with 15–50 µg of antibody. Only mono- and di-nucleosomes size DNA was used for construction of sequencing libraries. Sequencing libraries were prepared from two or more biological-replicate ChIP samples and from an input according to the instructions provided with the SOLiD Fragment Library Barcoding Kit (Life Technologies, Carlsbad, CA). The libraries were sequenced with SOLiD 4. The resulting reads were mapped using BioScope software (Life Technologies) with the default configuration, combined biological replicates, and analyzed using Homer (Heinz et al., 2010 (link)), CEAS (Shin et al., 2009 (link)) and R software programs. The mapping results are shown in Supplementary file 1A and were generated using IGV software (Robinson et al., 2011 (link)). The heatmap was generated using Java TreeView (http://jtreeview.sourceforge.net/).