BIOINFORMATICS APPLICATIONS NOTE
Vol. 28 no. 11 2012, pages 1530–1532
RNA-SeQC: RNA-seq metrics for quality control and
David S. DeLuca∗, Joshua Z. Levin, Andrey Sivachenko, Timothy Fennell,
Marc-Danie Nazaire, Chris Williams, Michael Reich, Wendy Winckler and Gad Getz∗
The Broad Institute of MIT and Harvard, Cambridge, MA, USA
Associate Editor: Ivo Hofacker
Advance Access publication April 25, 2012
Summary: RNA-seq, the application of next-generation sequencing
to RNA, provides transcriptome-wide characterization of cellular
activity. Assessment of sequencing performance and library quality
is critical to the interpretation of RNA-seq data, yet few tools exist
to address this issue. We introduce RNA-SeQC, a program which
provides key measures of data quality. These metrics include yield,
alignment and duplication rates; GC bias, rRNA content, regions of
alignment (exon, intron and intragenic), continuity of coverage, 3?/5?
bias and count of detectable transcripts, among others. The software
provides multi-sample evaluation of library construction protocols,
input materials and other experimental parameters. The modularity of
the software enables pipeline integration and the routine monitoring
of key measures of data quality such as the number of alignable
reads, duplication rates and rRNA contamination. RNA-SeQC allows
investigators to make informed decisions about sample inclusion
in downstream analysis. In summary, RNA-SeQC provides quality
control measures critical to experiment design, process optimization
and downstream computational analysis.
Availability and implementation: See www.genepattern.org to run
online, or www.broadinstitute.org/rna-seqc/ for a command line tool.
Supplementary information: Supplementary data are available at
Received on November 23, 2011; revised on March 8, 2012;
accepted on April 15, 2012
RNA-seq is a highly parallelized sequencing technology that allows
(Wang et al., 2009). As with all forms of parallelized sequencing,
significant computational processing is required to unlock transcript
abundance levels and other measures for biological interpretation
(Garber et al., 2011). However, prior to the calculation of
biologically relevant data such as transcript abundance, presence of
novel isoforms and genotype identity, it is necessary to evaluate the
performance of the RNA-seq experiment itself. Summary statistics
and quality control scores provide insight into inherently complex
data prior to downstream analysis.
Here we present RNA-SeQC, a metrics tool with application
to two domains: experiment design and process optimization;
and quality control prior to computational analysis. Metrics such
∗To whom correspondence should be addressed.
Fig. 1. Overview of the RNA-SeQC process. (a) RNA-SeQC will work
with one or more input samples to produce both a comparative summary
across samples as well as a more detailed report for each sample. (b) The
comparative summary report includes an extensive range of metrics (in
addition to those shown) as well as coverage plots. (c) For each sample,
additional reports quantify the coverage profile (variation, gaps, etc.) for
as duplication rate, rRNA abundance, alignment rates, coverage
continuity and correlation to reference expression profiles are highly
informative during selection of experiment conditions and library
construction methods (Levin et al., 2010). RNA-SeQC’s multi-
sample input feature allows for direct comparison across samples
(Fig. 1).Additionally, a single-sample mode can be used to monitor
samples on an ongoing basis to rapidly assess the quality of
a particular sequencing run, and to monitor and optimize these
measures in production over time and prior to downstream analysis.
RNA-SeQC provides a suite of experiment quality measures, many
of which are currently not provided by other available tools
RNA-SeQC provides three types of quality control metrics: Read
Counts, Coverage and Correlation. A list and description of
these metrics is shown below. RNA-SeQC is compatible with
any alignment method that produces a specification-conforming
BAM file (Li et al., 2009), with flags properly set. For additional
information, usage and software requirements, see the GenePattern
help document provided as Supplementary Material 1. Metrics
© The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
reports are provided in HTML for human consumption, as well as
tab-delimited text files for pipeline integration.
The following metrics are generated by counting reads with
particular characteristics. Rates are also provided, and are calculated
as either per total reads or per aligned reads. Since the BAM format
does support multiple alignments per read, this implementation
ignores any read flagged as not being a primary alignment.
• Total, unique and duplicate reads
• Mapped reads and mapped unique reads
• rRNA reads: counted in one of two modes: (i) interval
mode where an interval file defines the location in the given
alignment to which rRNA reads map; and (ii) BWA mode,
where an independent Burrows–Wheeler Aligner (Li and
Durbin, 2009) alignment to reference rRNA sequences is
• Transcript-annotated reads: intragenic (regions between
genes), intergenic (within genes), exonic and intronic.
These regions are defined in a user-specified GTF file
(Supplementary Material). GENCODE annotations (Harrow
et al., 2006) are used by default.
• Expression profile efficiency: the ratio of exon-mapped reads
to the total reads sequenced.
• Expressed transcripts: count of transcripts with reads ≥1.
library construction methods, the percentage of sense-derived
reads is given for each end of the read pair. Whereas a
non-strand-specific protocol would give values of 50%/50%,
strand-specific protocols typically yield 99%/1% or 1%/99%
for this metric.
The following metrics are based on coverage: the number of reads
that cover a given genomic position (in units of reads per base).
RNA-SeQC quantifies the uniformity of coverage with several
different metrics. To reflect the effect of expression level on these
metrics, we select genes from three categories: low, middle and high
expression genes (see Supplementary Material) and also report the
average of these metrics for each gene set.
• Mean coverage: the mean number of reads per base.
across all transcripts.
• 5?/3?Coverage: the mean per-base coverage for end regions
of RNAtranscripts. The length of the end region has a default
value of 100 base pairs.
• Gaps in coverage: a stretch of sequence of at least 5 base
pairs having zero coverage. Both the number of gaps as well
as the summed gap length across all transcripts in the set is
• Cumulative gap length: sum of gap lengths of all transcripts.
• Downsampling: to normalize data to a specific total read
count we enable an on-the-fly random reduction of reads to
reach a user-defined number. This is useful for comparing
certain statistics across datasets, e.g. gap metrics, which are
not otherwise adjusted for depth.
• GC bias: to assess effects of GC content on sequencing
performance, all coverage metrics are additionally reported
for three levels of transcript GC content: high, low and
moderate (see Supplementary Material for default threshold
• Coverage plots: plots of coverage level versus base index,
either for a single transcript or a set of transcripts.
One of the most valuable ways to interpret the performance of an
RNA-seq run is to compare the measured expression levels to a
reference (Levin et al., 2010). RNA-SeQC provides RPKM-based
estimation of expression levels (Mortazavi et al. 2008). When run
with multiple samples, RNA-SeQC creates a matrix of correlations
among all combinations, reporting the Spearman (rank based) and
Pearson (quantity based) correlation coefficients. Optionally, an
the different GC content stratifications to measure GC bias.
Implemented in Java, RNA-SeQC is platform independent and
requires no installation. For investigators who prefer a web interface
to a command-line tool, this software can be run using the
GenePattern web interface found at http://www.GenePattern.org
(Reich et al., 2006).
Within the RNA-SeQC software package, Read Count metrics
were implemented by inheriting from the ReadWalker class of
the GATK software package (McKenna et al., 2010). Transcript
annotations are bound to the walker in the RefGen format. This
format is created on-the-fly from a user-provided GTF file. The
program is designed to support the minimal GTF specification,
but the GTF format used by GENCODE (Harrow et al., 2006) is
recommended. For continuity of coverage calculations, the GATK’s
at a given position in the genomic alignment. Finally, ribosomal
RNA quantification is performed by realigning all reads to rRNA
reference sequences using the Burrows–Wheeler Aligner (Li and
Funding: Funded in part with Federal funds from the National
Health, Department of Health and Human under Contract No.
Conflict of Interest: none declared.
Garber,M. et al. (2011) Computational methods for transcriptome annotation and
quantification using RNA-seq. Nat. Meth., 8, 469–477.
D.S.DeLuca et al.
Harrow,J. et al. (2006) GENCODE: producing a reference annotation for ENCODE.
Genome Biol., 7 (Suppl. 1), S4.1–S4.9.
Levin,J.Z. et al. (2010) Comprehensive comparative analysis of strand-specific RNA
sequencing methods. Nat. Meth., 7, 709–715.
Li,H. and Durbin,R. (2009) Fast and accurate short read alignment with Burrows-
Wheeler transform. Bioinformatics, 25, 1754–1760.
Li,H. et al. (2009) The sequence alignment/map (SAM) format and SAMtools.
Bioinformatics, 25, 2078–2079.
McKenna,A. et al. (2010) The Genome Analysis Toolkit: a MapReduce framework
for analyzing next-generation DNA sequencing data. Genome Res., 20,
Mortazavi,A. et al. (2008) Mapping and quantifying mammalian transcriptomes by
RNA-Seq. Nat. Meth., 5, 621–628.
Reich,M. et al. (2006) GenePattern 2.0. Nat. Genet., 38, 500–501.
Wang,Z. et al. (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev.
Genet., 10, 57–63.