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CDNA hybrid capture improves transcriptome analysis on low-input and archived samples.

ABSTRACT: The use of massively parallel sequencing for studying RNA expression has greatly enhanced our understanding of the transcriptome through the myriad ways these data can be characterized. In particular, clinical samples provide important insights about RNA expression in health and disease, yet these studies can be complicated by RNA degradation that results from the use of formalin as a clinical preservative and by the limited amounts of RNA often available from these precious samples. In this study we describe the combined use of RNA sequencing with an exome capture selection step to enhance the yield of on-exon sequencing read data when compared with RNA sequencing alone. In particular, the exome capture step preserves the dynamic range of expression, permitting differential comparisons and validation of expressed mutations from limited and FFPE preserved samples, while reducing the data generation requirement. We conclude that cDNA hybrid capture has the potential to significantly improve transcriptome analysis from low-yield FFPE material.

SUBMITTER: Cabanski CR

PROVIDER: S-EPMC4078367 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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cDNA hybrid capture improves transcriptome analysis on low-input and archived samples.

Cabanski Christopher R CR Magrini Vincent V Griffith Malachi M Griffith Obi L OL McGrath Sean S Zhang Jin J Walker Jason J Ly Amy A Demeter Ryan R Fulton Robert S RS Pong Winnie W WW Gutmann David H DH Govindan Ramaswamy R Mardis Elaine R ER Maher Christopher A CA

The Journal of molecular diagnostics : JMD 20140509 4

The use of massively parallel sequencing for studying RNA expression has greatly enhanced our understanding of the transcriptome through the myriad ways these data can be characterized. In particular, clinical samples provide important insights about RNA expression in health and disease, yet these studies can be complicated by RNA degradation that results from the use of formalin as a clinical preservative and by the limited amounts of RNA often available from these precious samples. In this stu ...[more]

PMID: 24814956

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Project description:BackgroundCell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used.ResultsWe obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a 'gold standard' control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of the SMART-Seq v4 processed samples were similar to TruSeq (Spearman correlation > 0.8) despite using lower amount of input RNA. All RiboTag-IP samples had an increase in the intronic reads compared with the corresponding whole tissue, suggesting that the IP captures some immature mRNAs. The SMARTer-processed samples had a higher representation of ribosomal and non-coding RNAs leading to lower representation of protein coding mRNA. The enrichment or depletion of IP samples compared to corresponding input RNA was similar across all kits except for SMARTer kit.ConclusionRiboTag-seq can be performed successfully with as little as 250 pg of total RNA when using the SMART-Seq v4 kit and 4 ng when using the modified protocols of other library preparation kits. The SMART-Seq v4 and TruSeq kits resulted in the highest quality libraries. RiboTag IP RNA contains some immature transcripts.

Dataset Information

CDNA hybrid capture improves transcriptome analysis on low-input and archived samples.

Publications

cDNA hybrid capture improves transcriptome analysis on low-input and archived samples.

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