File S1 - In-Depth Characterization of microRNA Transcriptome in Melanoma

Contains: Figure S1. Flow chart for small RNA library construction, sequencing controls, false discovery rate (FDR) and clustering of melanoma specimens and cell lines. Flow chart shows small RNA capturing, amplification and multiplex sequencing (A). Unsupervised clustering of four technical replicates – cultured primary melanocytes from an individual with medium skin color (CMELM) demonstrated a nearly identical miRNA expression pattern between each control (B). Choosing the top-40 miRNAs as classifiers resulted in <10% FDR (C). Unsupervised clustering segregated the primary cutaneous melanoma (PCM) from normal skin (NS), common nevus (CN) and metastatic melanoma to skin (MMS) and lymph node (MMLN) hierarchical clustering of the samples using complete linkage and correlation-based distance (D). Moreover, cultured primary melanoma cell lines (WM35 and C32) clustered together and separated from metastatic cell lines (A375p and A375SM) andmelanocytes. Cultured melanocytes of light (CMELL), medium (CMELM) and dark (CMELD) skin color were segregated according to the melanin content. Figure S2. Comparison of the quality of small RNA library between FFPE primary cutaneous melanomas and primary melanoma cell lines. Both the electrophoresis summary and peak analysis show small RNA library constructed using archived FFPE melanomas (A), s130–s133, and using cell lines (B), WM 35 (EF 35) and WM278 (EF 278). Both FFPE specimens and cell lines showed a single sharp peak in the excepted range 144–150 bp, indicating intact captured small RNAs. Figure S3. Comparison of miRNA expression between NGS and qRT-PCR in discovery cohort. The expression levels of miR-211 (A), let-7i (B) and miR451a (C) were compared between disease groups: normal skin (NS), common nevus (CN), primary cutaneous melanoma (PCM), and metastatic melanoma to lymph node (MMLN) and to skin (MMS). The fold difference by NGS for every miRNA in a given sample was normalized per total miRNA sequence counts for that sample. The fold difference by qRT-PCR was expressed as RQ values for every specimen. Table S1. Illumina (Solexa) flow-cell sample description and barcode sequence used in NGS. Table S2. Clinicopathologic characteristics for validation cohort. Table S3. Pairwise statistical comparisons of miRNA levels to diagnostic groups by Tukey and non-parametric methods. Table S4. Predicted gene pathways and gene targets perturbed by deregulated miRNAs in melanoma. Table S5. Novel miRNA predicted folding, processed and compiled hairpin sequences. Table S6. Novel miRNA chromosomal loci and putative target genes. Table S7. Examples of differences in isomeric read counts of deregulated miRNAs in all specimens combined.
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