Molina, M. | Search bioRxiv

bioRxiv2019

Inherited duplications of PPP2R3B promote naevi and melanoma via a novel C21orf91-driven proliferative phenotype

The majority of the heredity of melanoma remains unexplained, however inherited copy number changes have not yet been systematically studied. The genetic environment is highly relevant to treatment stratification, and new gene discovery is therefore desirable. Using an unbiased whole genome screening approach for copy number we identify here a novel melanoma predisposing factor, familial duplications of gene PPP2R3B, encoding a regulatory unit of critical phosphatase PP2A. Significant correlation between expression of PPP2R3B in tumour tissue and survival in a large melanoma cohort was confirmed, and associated with a non-immunological expression profile. Mechanistically, construction and extensive characterization of a stable, inducible cellular model for PPP2R3B overexpression revealed induction of pigment cell switching towards proliferation and away from migration. Importantly, this was independent of the known microphthalmia-associated transcription factor (MITF)-controlled pigment cell phenotype switch, and was instead driven by uncharacterised gene C21orf91. Bioinformatic studies point to C21orf91as a novel target of MITF, and therefore a potential hub in the control of phenotype switching in melanoma. This study identifies novel germline copy number variants in PPP2R3B predisposing to melanocytic neoplasia, and uncovers a new potential therapeutic target C21orf91 in the control of pigment cell proliferation.

genomics↗

bioRxiv2019

Adapterama II: Universal amplicon sequencing on Illumina platforms (TaggiMatrix)

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. Most NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5 end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (eight forward primers + 12 reverse primers yield 8 x 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

genomics↗