Computational protocol: Next Generation Sequencing Analysis of Human Platelet PolyA+ mRNAs and rRNA Depleted Total RNA

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Protocol publication

[…] The cDNA obtained from the platelet polyA+ mRNA sample was shotgun sequenced (1×100 bp single read module) with the Illumina HiSeq 2000® instrument (Illumina, San Diego, CA, USA) by using a customer sequencing service (Eurofins MWG Operon, Ebersberg, Germany) which also included nebulization and end repair of cDNA, ligation of adaptors, gel purification, PCR amplification and library purification. The number of raw sequencing reads was 65,111,491. Filtering to remove bad quality bases and reads resulted in 58,155,680 reads (89.3%). These sequences were then mapped against the set of chromosomes of the Human Feb. 2009 (GRCh37/hg19) assembly. Initially, the mapping was conducted using the software TopHat 1.2.0 (http://tophat.cbcb.umd.edu). The post-processing of the mapping results was conducted using SamTools 0.1.12 a (http://samtools.sourceforge.net) and custom made Ruby 1.8.7 software. Bowtie http://bowtie-bio.sourceforge.net/ and bwa http://bio-bwa.sourceforge.net/ were used for aligning to de novo assembled transcripts and RefSeq mRNA respectively.RNA samples from three platelet donors were prepared for total RNA sequencing. For these samples, ribosomal RNA was depleted with Ribo-Zero (Epicentre, Madison, WI, USA) and strand specific barcoded RNA-sequencing libraries were prepared using ScriptSeq v2 (Epicentre) according to manufacturers instructions. The barcoded libraries were run on a single lane paired end 100 bp on a HiSeq2000® (Illumina, San Diego, CA, USA), which resulted in 153 million pass filter read pairs. QC data can be found in Figure S1 in . The TopHat2 software was used with the bowtie aligner. [...] de novo assembly of transcripts was performed using the Trinity RNA-Seq software (http://trinityrnaseq.sourceforge.net). […]

Pipeline specifications

Software tools TopHat, SAMtools, Bowtie, BWA, Trinity
Organisms Homo sapiens
Diseases Thrombosis