Preparation of Amplicon Libraries for Metabarcoding of Marine Eukaryotes Using Illumina MiSeq: The Dual-PCR Method.

This protocol details the preparation of multiplexed amplicon libraries for metabarcoding (amplicon-based) studies of microscopic marine eukaryotes. Metabarcoding studies, based on the amplification of a taxonomically informative marker from a collection of organisms or an environmental sample, can be performed to analyze biodiversity patterns or predator-prey interactions. For Metazoa, we use the mitochondrial cytochrome oxidase 1 (CO1) or the small ribosomal subunit (SSU) markers. Here, we describe a strategy for the preparation of multiplexed Illumina MiSeq libraries using a dual-PCR approach for the addition of index and adaptor sequences.

Tumor development, growth characteristics and spectrum of genetic aberrations in the TH-MYCN mouse model of neuroblastoma.

BACKGROUND: The TH-MYCN transgenic neuroblastoma model, with targeted MYCN expression to the developing neural crest, has been used to study neuroblastoma development and evaluate novel targeted tumor therapies. METHODS: We followed tumor development in 395 TH-MYCN (129X1/SvJ) mice (125 negative, 206 hemizygous and 64 homozygous mice) by abdominal palpations up to 40 weeks of age. DNA sequencing of MYCN in the original plasmid construct and mouse genomic DNA was done to verify the accuracy. Copy number analysis with Affymetrix® Mouse Diversity Genotyping Arrays was used to characterize acquired genetic aberrations. RESULTS: DNA sequencing confirmed presence of human MYCN cDNA in genomic TH-MYCN DNA corresponding to the original plasmid construct. Tumor incidence and growth correlated significantly to transgene status with event-free survival for hemizygous mice at 50%, and 0% for homozygous mice. Hemizygous mice developed tumors at 5.6-19 weeks (median 9.1) and homozygous mice at 4.0-6.9 weeks (5.4). The mean treatment window, time from palpable tumor to sacrifice, for hemizygous and homozygous mice was 15 and 5.2 days, respectively. Hemizygous mice developing tumors as early as homozygous mice had a longer treatment window. Age at tumor development did not influence treatment window for hemizygous mice, whereas treatment window in homozygous mice decreased significantly with increasing age. Seven out of 10 analysed tumors had a flat DNA profile with neither segmental nor numerical chromosomal aberrations. Only three tumors from hemizygous mice showed acquired genetic features with one or more numerical aberrations. Of these, one event corresponded to gain on the mouse equivalent of human chromosome 17. CONCLUSION: Hemizygous and homozygous TH-MYCN mice have significantly different neuroblastoma incidence, tumor growth characteristics and treatment windows but overlap in age at tumor development making correct early genotyping essential to evaluate therapeutic interventions. Contrasting previous studies, our data show that TH-MYCN tumors have few genetic aberrations.