Computational protocol: Development and characterization of a human orthotopic neuroblastoma xenograft

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Protocol publication

[…] Whole-genome sequencing (WGS) and SNP or gene expression profiling by array were performed as previously described (, ). For WGS, paired-end sequencing was performed using the Illumina GAIIx or HighSeq platform with 100 bp read length. The WGS data are deposited at the European Bioinformatics Institute (EBI) with accession number EGAS00001000256. WGS mapping, coverage and quality assessment, single nucleotide variation (SNV)/indel detection, tier annotation for sequence mutations, prediction of deleterious effects of missense mutations, and identification of loss of heterozygosity (LOH) have been described previously () Structural variations (SVs) were analyzed using CREST and annotated as previously described (; ). The reference human genome assembly NCBI Build 36 was used for mapping 5 samples (SJRHB001–005) and Build 37 was used for mapping other samples. Genomic coordinates for variations in samples mapped to Build 36 were lifted over to Build 37. Copy number variations (CNVs) were identified by evaluating the difference of read depth for each tumor and its matching normal using a novel algorithm, COpy Number SEgmentation by Regression Tree In Next-Gen sequencing (CONSERTING).For whole exome sequencing (WES), DNA libraries were prepared from 1 μg of whole genome amplified (WGA) material from matched samples using the Illumnia TruSeq DNA library prep kit following the recommended manufacturer’s protocol. Libraries were analyzed on an Agilent Bioanalyzer to inspect quality of each library construction. Germline and diagnostic library samples were independently pooled and applied for exome capture using the Illumina TruSeq Exome Enrichment kit as described by the manufacturer. Captured libraries were then clustered on the Illumina c-bot and were sequenced on an Illumina HiSeq 2000 platform with 100 base pair end multiplexed reads at an equivalent of 3 samples per lane.For enrichment and validation of the regions containing putative alterations, genomic coordinates of the putative WGS targets were used to order either Nimbelgen Seqcap EZ solution bait sets (Roche), or Nimblegen Seqcap 2.1M arrays (Roche). The library construction and target enrichment was performed per manufacturer’s instructions (Roche) using repli-G (Qiagen) WGA DNA. Enriched targets were sequenced on the illumina platform using paired end 100 cycle sequencing. The resulting data was converted to FASTQ files using CASAVA 1.8.2 (Illumina), and mapped with BWA prior to pipeline analysis.Putative SNVs from exon sequencing were validated by NGS amplicon sequencing. Briefly, primers were designed to genomic regions (hg19) flanking the SNV no closer than 100 base pairs to the detected SNV. PCR was performed using 20–30 ng of whole genome amplified (WGA) DNA from each patient sample. DNA from the tumor (diagnostic sample) and a matched germline sample were used for each primer set to confirm the presence of the SNV in the diagnostic sample. Standard PCR was performed in 25 μl reactions using Accuprime GC-rich DNA polymerase (Invitrogen) with the following parameters: 95 °C for 3 min, 35 cycles of 95 °C for 30 s, 65 °C for 30 s, 72 °C for 1 min followed by a 72 °C 10 min extension with cooling to 4 °C. All PCR amplicons were checked on a 2% E-gel (Invitrogen) to ensure single amplified products.PCR amplified products were collected into diagnostic or germ-line pools and purified using a Qiagen PCR purification kit. One nanogram of pooled sample was treated for sequencing using the Nextera XT kit (Illumina) following the manufacturer’s protocol. Samples were normalized and denatured prior to the MiSeq run using the 2 ×150 base pair run (version 1) reagent kit. […]

Pipeline specifications

Software tools CONSERTING, BaseSpace, BWA
Applications WGS analysis, WES analysis
Organisms Mus musculus, Homo sapiens
Diseases Neoplasms, Neuroblastoma
Chemicals Catecholamines