Tumor Sequencing
A pathologist with specific expertise in thyroid tumors (N.M.Q.)
examined the hematoxylin and eosin-stained tumor slides to identify
representative areas of tumor suitable for molecular testing. DNA and
RNA were extracted from FFPE tumor specimens according to the
manufacturers’ protocols utilizing the QIAamp DNA FFPE Tissue Kit
(Qiagen) and the RecoverAll Total Nucleic Acid Isolation Kit for FFPE
(Ambion), respectively. DNA and RNA were quantified by Qubit (Thermo
Fisher Scientific), with RNA undergoing an additional quality assessment
by the quantitative PCR assay PreSeq (ArcherDx).
Of the original 40 cases selected for sequencing, 4 cases were excluded
due to insufficient quantity and/or quality of nucleic acids. Therefore,
36 tumor samples with adequate quantity and quality of nucleic acids for
sequencing were utilized for NGS library preparation. DNA NGS libraries
were generated using a custom hybridization capture panel
(NimbleGen/Roche) designed to capture all coding exons of 124 genes
associated with pediatric solid tumors as well as the TERT gene
promoter 20. RNA NGS
libraries were generated using the Oncology Research Panel (ArcherDx)
that utilizes Anchored Multiplex PCR chemistry to capture gene fusion
events involving 75 recurrently rearranged cancer genes21. NGS libraries were
sequenced 4- to 6-plex on an Illumina MiSeq using V2 and V3 chemistry,
respectively, and de-mulitplexed using bcl2fastq v2.18.0.12 (Illumina),
achieving approximately 2.2-4.4 million sequencing reads per sample. The
resulting DNA FASTQ files were analyzed using a custom DNA
bioinformatics pipeline that includes alignment by BWA v0.7.1222 (Wellcome Trust
Sanger Institute) and deduplication and QC analysis by Picard v2.9.223. Variant calling was
completed by NextGENe v2.4.1.2 (SoftGenetics) and Platypus v0.8.124 (Wellcome Trust
Centre for Human Genetics) for the detection of SNVs and short indels,
with variant annotation by VEP v.8925 (Ensembl). Copy
number analysis was performed using CNVkit v0.9.326. CNVkit performs
circular binary segmentation on the normalized GC and target capture
density bias corrected log2 difference of binned read depths, using
on-target (average bin-size = 300bp) and off-target reads (average
bin-size = 500,000bp), between the tumor sample and reference
pooled-normal peripheral blood samples to identify genome-wide regions
of CNVs. Gene-level deletions and amplifications were then derived using
these copy number segments. RNA FASTQ files were analyzed by the Archer
Analysis suite v4.1.0.6 (ArcherDx) with additional custom wrapper and
text-processing scripts for the detection of gene fusion events.