Computational protocol: Spatial and temporal homogeneity of driver mutations in diffuse intrinsic pontine glioma

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Protocol publication

[…] Genomic DNA was extracted from multiple post-mortem samples patient using the standard extraction methods as described by Qiagen. Nextera Rapid Capture Exome kit was used to prepare the paired-end libraries according to the manufacturer's instructions using on average 36 ng of total starting genomic DNA. Sequencing was performed on Illumina HiSeq 2000 using rapid-run mode with 100 bp paired-end reads. Next, adaptor sequences were removed; reads were trimmed for quality using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). An in-house program was used to ensure the presence of exclusively paired-reads to be used in further steps of the analysis. We next aligned the reads using Burrows-Wheeler Aligner (BWA) 0.7.7 to hg19 as reference genome. Indel realignment was performed using the Genome Analysis Toolkit (GATK) (http://www.broadinstitute.org/gsa/wiki/). We next marked the duplicate reads using Picard (http://picard.sourceforge.net/) and excluded them from further analyses as previously described. The coverage of consensus coding sequence (CCDS) bases was assessed using GATK. The average coverage over all the samples was 70 × . The majority of samples had >90% of CCDS bases covered by at least 10 reads and >83% of CCDS bases covered by at least 20 reads.We called SNVs and short indels using SAMtools (http://samtools.sourceforge.net/) mpileup with the extended base alignment quality adjustment (–E). Next we filtered them for quality so that at least 10% of reads supporting each variant call. We used both ANNOVAR and in-house tools to annotate the variants and to identify whether these variants affect protein-coding sequence and if they had previously been observed in datasets including the 1,000 Genomes Project data set (November 2011), the National Heart, Lung and Blood Institute (NHLBI) Grand Opportunity (GO) exomes or in ∼3,000 exomes previously sequenced at our center. Results of whole exome sequencing are summarized in and presented for individual patients as follows: DIPG1 in ; DIPG2 in ; DIPG3 in ; DIPG4 in ; DIPG5 in ; DIPG6 in ; DIPG7 in ; DIPG8 in ; DIPG9 in . […]

Pipeline specifications

Software tools FASTX-Toolkit, BWA, GATK, Picard, SAMtools, ANNOVAR
Applications WES analysis, Nucleotide sequence alignment
Organisms Homo sapiens
Diseases Brain Neoplasms, Glioma, Neoplasms