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Evaluation of sequencing approaches for high-throughput toxicogenomics (SOT)
Citation:
Thomas, R., A. Karmaus, P. Kothiya, AND M. Martin. Evaluation of sequencing approaches for high-throughput toxicogenomics (SOT). Presented at Society of Toxicology 55th Annual Meeting, New Orleans, LA, March 13 - 17, 2016. https://doi.org/10.23645/epacomptox.5155975
Impact/Purpose:
This abstract presents work that evaluates three sequencing platforms for high-throughput toxicogenomics demonstrating the ability to measure whole-genome transcript levels with good technical reproducibility and showing promise for the integration of toxicogenomics into high-throughput screening.
Description:
Whole-genome in vitro transcriptomics has shown the capability to identify mechanisms of action and estimates of potency for chemical-mediated effects in a toxicological framework, but with limited throughput and high cost. We present the evaluation of three toxicogenomics platforms for potential application to high-throughput screening: 1. TempO-Seq utilizing custom designed paired probes per gene; 2. Targeted sequencing (TSQ) utilizing Illumina’s TruSeq RNA Access Library Prep Kit containing tiled exon-specific probe sets; 3. Low coverage whole transcriptome sequencing (LSQ) using Illumina’s TruSeq Stranded mRNA Kit. Each platform was required to cover the ~20,000 genes of the full transcriptome, operate directly with cell lysates, and be automatable with 384-well plates. Technical reproducibility was assessed using MAQC control RNA samples A and B, while functional utility for chemical screening was evaluated using six treatments at a single concentration after 6 hr in MCF7 breast cancer cells: 10 µM chlorpromazine, 10 µM ciclopriox, 10 µM genistein, 100 nM sirolimus, 1 µM tanespimycin, and 1 µM trichostatin A. All RNA samples and chemical treatments were run with 5 technical replicates. The three platforms achieved different read depths, with the TempO-Seq having ~34M mapped reads per sample, while TSQ and LSQ averaged 20M and 11M aligned reads per sample, respectively. Inter-replicate correlation averaged ≥0.95 for raw log2 expression values in all three platforms across all samples. When the ratio of MAQC samples A:B was correlated between the technologies and the reference MAQC-III Illumina results, r2 values of 0.83 for LSQ, 0.74 for TSQ, and 0.75 for TempO-Seq were observed, suggesting good technical reproducibility for each sequencing platform. When chemically-treated samples were evaluated, the inter-replicate and cross-technology correlations of fold-change values were significantly reduced. Bland-Altman plots revealed that genes with low read counts accounted for the greatest variability in fold-change space. Application of a minimum read-count cutoff was necessary to achieve good concordance. Finally, connectivity map (CMAP) analysis was conducted to evaluate the ability of each platform to identify modes-of-action in the chemically-treated samples. TempOSeq showed the best concordance with mechanistically similar chemical treatments; however, this may be due to the increased read depth associated with the platform. In summary, the three toxicogenomics platforms had the ability to measure whole-genome transcript levels with good technical reproducibility and show promise for the integration of toxicogenomics into high-throughput screening.This abstract does not necessarily reflect US EPA policy.
URLs/Downloads:
DOI: Evaluation of sequencing approaches for high-throughput toxicogenomics (SOT)
ALKARMAUS SOT2016 HTTSEQ POSTER V3 FINAL.PDF (PDF, NA pp, 1303.412 KB, about PDF)