Transcriptome Analysis of FFPE and Frozen Samples

RNA-seq was performed on one fresh-frozen sample from P1 and one FFPE sample from P2. RNA was extracted using Promega Maxwell 16 MDX and RNA integrity was verified using the Agilent Bioanalyzer 2100 (Agilent Technologies). cDNA was synthesized from total RNA using Superscript III (Invitrogen). Sequencing was performed using Illumina HiSeq2500 in 2 × 75 bp paired-end mode, and generated an average of 45 million reads per sample. All reads were independently aligned with STAR_2.4.0f for sequence alignment against the human genome build hg19, downloaded via the UCSC genome browser (http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/), and SAMTOOLS v0.1.19 for sorting and indexing reads (Li et al. 2009 (link)). For fusion analysis, we used STAR-fusion (STAR-Fusion_v0.5.1) and FusionCatcher (v0.99.3e). Fusions with significant support of junction reads and spanning pairs were selected and manually reviewed.

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Cyrta J., Rosiene J., Bareja R., Kudman S., Al Zoughbi W., Motanagh S., Wilkes D.C., Eng K., Zhang T., Sticca E., Mathew S., Rubin M.A., Sboner A., Elemento O., Rubin B.P., Imielinski M, & Mosquera J.M. (2022). Whole-genome characterization of myoepithelial carcinomas of the soft tissue. Cold Spring Harbor Molecular Case Studies, 8(7), a006227.

Publication 2022

Maxwell 16 MDX Agilent Bioanalyzer 2100 Superscript III HiSeq2500

Cdna Frozen Genome Human genome Promega Rna seq Sequence alignment

Corresponding Organization :

Other organizations : Cornell University, SUNY Downstate Health Sciences University, Cleveland Clinic, New York Genome Center

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Variable analysis

independent variables

RNA extraction method (Promega Maxwell 16 MDX)
Sample type (fresh-frozen from P1, FFPE from P2)

dependent variables

RNA integrity (verified using Agilent Bioanalyzer 2100)
Sequencing reads (average of 45 million reads per sample generated using Illumina HiSeq2500 in 2 × 75 bp paired-end mode)
Sequence alignment (using STAR_2.4.0f and SAMTOOLS v0.1.19)
Fusion analysis (using STAR-fusion and FusionCatcher)

control variables

CDNA synthesis (using Superscript III, Invitrogen)
Human genome build (hg19, downloaded from UCSC genome browser)

positive controls

None specified

negative controls

None specified

Annotations

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