Computational protocol: Analysis of the Expression of Repetitive DNA Elements in Osteosarcoma

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Protocol publication

[…] Forty to fifty milligrams of bone sample was grinded with nitrogen by pestle and mortar into powder and pre-treated with trizol. We used RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia CA, USA) to extract total RNA from bone tissue according to the manufacturer's protocol. Extracted RNA was dissolved in RNase free water and stored at −80°C. Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit (Agilent Technologies Inc., CA, USA) were used to measure the quality of total RNA.By applying Ovation RNA-Seq System V2 (NuGen, Emeryville, CA, USA), Fifty nanograms of total RNA was amplified. The collected cDNAs were pooled in equal amount and we used the pool to prepare the DNA fragment library by using SOLiD System chemistry (Life Technologies Corp, Carlsbad, CA, USA). SOLiD 5500W platform and DNA sequencing chemistry (Life Technologies Corp., Carlsbad, CA, USA) were applied for sequencing. By using Maxmapper algorithm implemented in the Lifescope software (Life Technologies, Ltd), Raw reads (75 bp) were color-space mapped to the human genome hg19 reference using. It was permitted to map multiple locations. The mapping confidence was more than 90 because the quality threshold was set to 10. Reads were filtered out if the score was < 10. Mapping quality was at an average of 30. The RepEnrich pipeline with RepeatMasker hg19 library as reference was used for obtaining read counts based on reads aligning to repeated element loci. [...] For statistical analysis, edgeR (empirical analysis of DGE-digital gene expression in R) package for R was used (Robinson et al., ). EdgeR is a bioconductor package in R used to analyse the differential expression of digital gene expression data. It was developed for RNA-seq or other counts data. It provides methods to analyse the differential expression by using the negative binomial distribution and a shrinkage estimator for the distribution's variance. The Package performs sample comparison and it can adjust the P-value to overcome problems of multiple testing (Li et al., ; McCarthy et al., ; Anders et al., ). EdgeR package uses Benjamini-Hochberg procedure to control the false discovery rate (FDR) (Benjamini and Hochberg, ). […]

Pipeline specifications

Software tools LifeScope, RepEnrich, RepeatMasker, edgeR
Applications RNA-seq analysis, DGE analysis
Organisms Homo sapiens
Diseases Bone Neoplasms, Neoplasms, Osteosarcoma