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Protocol publication

[…] oblem to assign reads to a particular isoform if there are many transcript variants with overlaps between them. Very high coverage figures are needed for satisfactory results. This is one of the reasons why RNA-Seq with low coverage has many of the same limitations as other RNA expression analysis pipelines., Obviously, mapping of reads against the human genome and also mapping against the human exome both rely on the accuracy of gene and transcript annotations. In order to fully characterize the platelet transcriptome without reference to previous results, including the possibility to detect and fully characterize novel transcripts, we also performed a de novo assembly of transcripts using Trinity RNA-Seq software (http://trinityrnaseq.sourceforge.net/). This software will extract full-length transcripts for alternatively spliced isoforms based on the generation and analysis of de Bruijn graphs. RSEM software with the bowtie aligner (http://bowtie-bio.sourceforge.net/) was used for mapping the RNA-Seq reads back to the reported transcripts for abundance estimation. Identification of the transcripts was achieved by Blat and BLAST searches using the UCSC Browser and the NCBI Genome databases, respectively. These data fully supported our results obtained by mapping the reads to the human genome and exome, respectively, using gtf.guided assembly. However, even if transcript abundance figures agreed only the most abundant transcripts could be reliably reconstructed by the de novo assembly approach; presumably due to insufficient amounts of reads that were available., When we started this study there was no published RNA-Seq data on platelet gene expression although microarray based as well as SAGE and real-time PCR methods have been used in the past. However, two studies using RNA-Seq by NGS were published […]

Pipeline specifications

Software tools Trinity, RSEM, Bowtie, BLAT
Organisms Homo sapiens
Diseases Embolism and Thrombosis