Computational protocol: Feasibility of implementing molecular-guided therapy for the treatment of patients with relapsed or refractory neuroblastoma

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Protocol publication

[…] RNA sequencing was performed using 1.0 µg of total RNA quantified via Nanodrop (Thermo Scientific, Pittsburgh, PA). A sequencing library was prepared with Illumina's Truseq RNA Sample Preparation Kit v2 (Illumina Inc, San Diego, CA) following the manufacturer's protocol. In brief, poly-A containing mRNA molecules were purified using poly-T oligo attached magnetic beads. The mRNA was then thermally fragmented and converted to double-stranded cDNA. The cDNA fragments were end-repaired, a single “A” nucleotide was incorporated, sequencing adapters were ligated, and fragments were enriched with 15 cycles of PCR. Final PCR-enriched fragments were validated on a 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) and quantified by qPCR using Kapa's Library Quantification Kit (Kapa Biosystems, Woburn, MA) on the 7900HT (Applied Biosystems, Foster City, CA). The final library was sequenced by 50 bp paired-end sequencing on a HiSeq2000 (Illumina, San Diego, CA).Raw reads passing Illumina quality filters were converted to FASTQ format in Phred33 scale with CASAVA 1.8.3. RNA-Seq reads were aligned with TopHat (v2.0.8) which first utilizes Bowtie (v2.1.0.0) to map reads with “splice-aware” alignments to the Homo Sapiens build GRCh37 from Ensembl . To estimate the library fragment size for TopHat, we initially mapped a subset of 1 million reads with bwa (v0.6.1) to the human genome, followed by picard version 1.80 module CollectInsertSizeMetrics and provided these values to TopHat options “–mate-inner-dist 87 –mate-std-dev 86.” Additional TopHat flags utilized were –transcriptome-index (to Ensembl GRCh37.70), –no-coverage-search, –b2-sensitive and –keep-fasta-order. Next, we calculated gene expression values expressed as fragments per kilobase pair of exon per million fragments mapped using cufflinks version 2.1.1 . We used the –GTF option in cufflinks to annotate to human gene models GRCh37.70. Additionally we used the –multi-read-correct and –frag-bias-correct options in cufflinks and masked tRNAs, rRNAs, and mtRNAs as suggested in the cufflinks documentation. […]

Pipeline specifications

Software tools BaseSpace, TopHat, Bowtie, BWA, Picard, Cufflinks
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Neoplasms, Neuroblastoma