RNA-seq is an effective method for studying the transcriptome, but it can be difficult to apply to scarce or degraded RNA from fixed clinical samples, rare cell populations or cadavers. Recent studies have proposed several methods for RNA-seq of low-quality and/or low-quantity samples, but the relative merits of these methods have not been systematically analyzed. Here we compare five such methods using metrics relevant to transcriptome annotation, transcript discovery and gene expression. Using a single human RNA sample, we constructed and sequenced ten libraries with these methods and compared them against two control libraries. We found that the RNase H method performed best for chemically fragmented, low-quality RNA, and we confirmed this through analysis of actual degraded samples. RNase H can even effectively replace oligo(dT)-based methods for standard RNA-seq. SMART and NuGEN had distinct strengths for measuring low-quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development.
At a glance
- Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69, 1408–1412 (1972). &
- Genomewide characterization of non-polyadenylated RNAs. Genome Biol. 12, R16 (2011). , , , &
- mRNA-Seq whole-transcriptome analysis of a single cell. Nat. Methods 6, 377–382 (2009). et al.
- Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777–782 (2012). et al.
- Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res. 21, 1160–1167 (2011). et al.
- Methods for depleting RNA from nucleic acid samples. US patent application 20110111409 (2011). &
- Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue. PLoS ONE 7, e42882 (2012). , &
- An RNA-Seq strategy to detect the complete coding and non-coding transcriptome including full-length imprinted macro ncRNAs. PLoS ONE 6, e27288 (2011). et al.
- Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq. Nucleic Acids Res. 39, e140 (2011). et al.
- Whole-transcriptome RNAseq analysis from minute amount of total RNA. Nucleic Acids Res. 39, e120 (2011). , , &
- Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat. Methods 7, 709–715 (2010). et al.
- RNA-SeQC: RNA-seq metrics for quality control and process optimization. Bioinformatics 28, 1530–1532 (2012). et al.
- Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. Genes Dev. 2, 754–765 (1988). &
- Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15 (2002). et al.
- Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol. 12, R18 (2011). et al.
- Characterizing the mouse ES cell transcriptome with Illumina sequencing. Genomics 92, 187–194 (2008). , , &
- Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes. Genome Biol. 13, R23 (2012). et al.
- Simplified and versatile method for isolation of high-quality RNA from pancreas. Biotechniques 52, 332–334 (2012). , , , &
- Two methods for full-length RNA sequencing for low quantities of cells and single cells. Proc. Natl. Acad. Sci. USA 110, 594–599 (2013). et al.
- Improving RNA-Seq expression estimates by correcting for fragment bias. Genome Biol. 12, R22 (2011). , , , &
- Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009). &
- Clones of human ribosomal DNA containing the complete 18 S-rRNA and 28 S-rRNA genes. Characterization, a detailed map of the human ribosomal transcription unit and diversity among clones. Biochem. J. 246, 519–527 (1987). et al.
- TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009). , &
- The UCSC Genome Browser database: extensions and updates 2011. Nucleic Acids Res. 40, D918–D923 (2012). et al.
- RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011). &
- Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009). , , &
- ggplot2: Elegant Graphics for Data Analysis (Use R!) (Springer, New York, 2009).
- Supplementary Text and Figures (i) (47 MB)
Supplementary Figures 1–7
- Supplementary Text and Figures (ii) (21 MB)
Supplementary Figures 8 and 9
- Supplementary Text and Figures (iii) (782 KB)
Supplementary Tables 1–5 and 7 and Supplementary Notes 1–5
- Supplementary Table 6 (20 KB)
RNase H oligonucleotide sequences