Computational protocol: Potential pitfalls of mass spectrometry to uncover mutations in childhood soft tissue sarcoma: A report from the Children’s Oncology Group

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Protocol publication

[…] To validate the potential mutations identified by MassARRAY, we performed deep sequencing of the genomic regions containing the mutations. One case (PASNLT) could not be included in the validation set due to insufficient remaining DNA. Primers were designed to amplify approximately 150 bp segments (range 118 bp–329 bp) of DNA surrounding the mutations using SeqBuilder (DNASTAR, Madison, WI) and Primer-BLAST software (). For each reaction, 1 ng of input DNA underwent 35 cycles of PCR (20 sec denature, 15 sec anneal, 15 sec extend) using the KAPA HiFi (high fidelity) Hot Start DNA polymerase (KAPA Biosystems, Wilmington, MA). Each reaction was carried out independently, as opposed to being multiplexed. The PCR products were loaded and run on a 0.8% agarose, 0.0005% ethidium bromide gel, the bands of interest cut out with a clean, sterile razorblade on a long-wavelength (365 nm) UV box (3UV Transilluminator, UVP, Upland, CA), and extracted using the QIAquick Gel Extraction Kit (Qiagen), according to manufacturer’s specifications. Library preparation and sequencing were carried out at the McDermott Next Generation Sequencing Core. Briefly, DNA concentration was measured using a Qubit Fluorometer (Life Technologies). Library preparation was carried out using the KAPA HTP Library Preparation Kit (KAPA Biosystems) with Illumina TruSeq adapters (Illumina, San Diego, CA) at a 1:2 dilution and seven cycles of PCR. The library was quantified using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and the samples were sequenced on a HiSeq 2500 (Illumina) with 100 bp paired-end reads.To assess the detection limits of this assay, we studied two variants in NRAS, one each found in either the RD or Rh30 rhabdomyosarcoma cell line. We serially diluted the RD DNA in Rh30 DNA and PCR amplified approximately 200 bp segments of DNA surrounding each sequence variant, using the same methods as described above. The products from the two reactions were gel purified, combined, and sent to the NGS Core for sequencing. To approximate the effect of formalin fixation, RD and Rh30 cells were pelleted into two tubes per cell line (1 × 106 cells per tube) and either flash frozen in liquid nitrogen or fixed with 4% paraformaldehyde (PFA) for 15 minutes, then washed three times in PBS, prior to DNA extraction. [...] All raw sequencing reads were mapped to the human reference genome (hg19) using the Burrows-Wheeler Aligner. Picard was used to convert SAM files to BAM files and remove PCR duplicates (http://broadinstitute.github.io/picard/). Our analysis pipeline also realigned the sequence reads to obtain more accurate quality scores and then recalibrated the base qualities by GATK before variant calling. Variant calling was done by directly measuring the number of supporting reads for A, T, G and C on the genomic loci containing MassARRAY-identified mutations. Our computational pipeline compared variants against common polymorphisms present in the dbSNP 138 and 1000 Genomes databases, as well as COSMIC version 73, a database of cancer somatic mutations. Several gene transcript annotation databases (Consensus CDS, RefSeq, Ensembl and UCSC) were used to determine amino acid changes. All BAM files and programming scripts are available upon request. […]

Pipeline specifications

Software tools SeqBuilder Pro, Primer-BLAST, BWA, Picard, GATK
Applications WES analysis, qPCR
Organisms Homo sapiens
Diseases Neoplasms, Sarcoma