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Simul-seq: combined DNA and RNA sequencing for whole-genome and transcriptome profiling.

ABSTRACT: Paired DNA and RNA profiling is increasingly employed in genomics research to uncover molecular mechanisms of disease and to explore personal genotype and phenotype correlations. Here, we introduce Simul-seq, a technique for the production of high-quality whole-genome and transcriptome sequencing libraries from small quantities of cells or tissues. We apply the method to laser-capture-microdissected esophageal adenocarcinoma tissue, revealing a highly aneuploid tumor genome with extensive blocks of increased homozygosity and corresponding increases in allele-specific expression. Among this widespread allele-specific expression, we identify germline polymorphisms that are associated with response to cancer therapies. We further leverage this integrative data to uncover expressed mutations in several known cancer genes as well as a recurrent mutation in the motor domain of KIF3B that significantly affects kinesin-microtubule interactions. Simul-seq provides a new streamlined approach for generating comprehensive genome and transcriptome profiles from limited quantities of clinically relevant samples.

SUBMITTER: Reuter JA

PROVIDER: S-EPMC5734913 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Simul-seq: combined DNA and RNA sequencing for whole-genome and transcriptome profiling.

Reuter Jason A JA Spacek Damek V DV Pai Reetesh K RK Snyder Michael P MP

Nature methods 20161010 11

Paired DNA and RNA profiling is increasingly employed in genomics research to uncover molecular mechanisms of disease and to explore personal genotype and phenotype correlations. Here, we introduce Simul-seq, a technique for the production of high-quality whole-genome and transcriptome sequencing libraries from small quantities of cells or tissues. We apply the method to laser-capture-microdissected esophageal adenocarcinoma tissue, revealing a highly aneuploid tumor genome with extensive blocks ...[more]

PMID: 27723755

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Project description:RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN's Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates.We found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P?<?0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison.Transcriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging.

Project description:BackgroundFormalin-fixed paraffin-embedded (FFPE) tissue samples are routinely archived in the course of patient care and can be linked to clinical outcomes with long-term follow-up. However, FFPE tissues have degraded RNA which poses challenges for analyzing gene expression. Next-generation sequencing (NGS) is rapidly becoming accepted as an effective tool for measuring gene expressions for research and clinical use. However, the feasibility of NGS has not been firmly established when using FFPE tissue.ResultsWe optimized strategies for whole transcriptome sequencing (RNA-seq) using FFPE tissue. Ribosomal RNA (rRNA) was successfully depleted by competitive hybridization using the Ribo-zero™ Kit (Epicentre Biotechnologies), and rRNA sequence content was less than one percent for each library. Gene expression measured by FFPE RNA-seq was compared to two different standards: RNA-seq from fresh frozen (FF) tissue and quantitative PCR (qPCR). Both FF and FFPE tumors were sequenced on an Illumina Genome Analyzer IIX with an average of 10 million reads. The distribution of FPKMs (fragments per kilobase of exon per million fragments mapped) and number of detected genes were similar between FFPE and FF. RNA-seq expressions from FF and FFPE samples from the same renal cell carcinoma (RCC) correlated highly (r = 0.919 for tumor 1 and r = 0.954 for tumor 2). On hierarchical cluster analysis, samples clustered by patient identity rather than method of preservation. TaqMan qPCR of 424 RCC-related genes correlated highly with FFPE RNA-seq expressions (r = 0.775 for FFPE tumor 1, r = 0.803 for FFPE tumor 2). Expression fold changes were considered, to assess biologic relevance of gene expressions. Expression fold changes between FFPE tumors (tumor 1/tumor 2) correlated well when comparing qPCR and RNA-seq (r = 0.890). Expression fold changes between tumors from different risk groups (our high risk RCC/The Cancer Genome Atlas, TCGA, low risk RCC) also correlated well when comparing RNA-seq from FF and FFPE tumors (r = 0.887).ConclusionsFFPE RNA-seq provides reliable genes expression data, comparable to that obtained from fresh frozen tissue. It represents a useful tool for discovery and validation of biomarkers.

Dataset Information

Simul-seq: combined DNA and RNA sequencing for whole-genome and transcriptome profiling.

Publications

Simul-seq: combined DNA and RNA sequencing for whole-genome and transcriptome profiling.

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