Biases in small RNA deep sequencing data

Abstract

High-throughput RNA sequencing (RNA-seq) is considered a powerful tool for novel gene discovery and fine-tuned transcriptional profiling. The digital nature of RNA-seq is also believed to simplify meta-analysis and to reduce background noise associated with hybridization-based approaches. The development of multiplex sequencing enables efficient and economic parallel analysis of gene expression. In addition, RNA-seq is of particular value when low RNA expression or modest changes between samples are monitored. However, recent data uncovered severe bias in the sequencing of small non-protein coding RNA (small RNA-seq or sRNA-seq), such that the expression levels of some RNAs appeared to be artificially enhanced and others diminished or even undetectable. The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies and exemplify sources of bias in deep sequencing. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies. Given the central importance of RNA-seq to molecular biology and personalized medicine, we review recent findings that challenge small non-protein coding RNA-seq data and suggest approaches and precautions to overcome or minimize bias.

Figure 1.

Illustration of the steps involved in cDNA construction, including potential sources of bias. (A) The starting pool of non-protein coding RNAs with different 5′- and 3′-end modifications schematically indicated by different line types. Abbreviations for the various modifications: OH: hydroxyl, OPO₃: 2′-3′-cyclic phosphate, ppp: triphosphate, p: monophosphate, cap: cap, and 2′-OCH₃: 2′-O-methyl. (B) The left panel depicts different enzymatic pre-treatments prior to RNA 5′-end ligation to enrich for different RNA subtypes. From left to right: RNA without any pretreatment (5′-adapter ligation); RNA pretreated with tobacco acid phosphatase (TAP); RNA pretreated with Terminator™ 5′-phosphate-dependent exonuclease and TAP (Terminator 5′-exo; TAP); RNA treated with T4 polynucleotide kinase (T4 PNK). RNA classes accessible for adapter ligation after the respective 5′-end pretreatments are schematically represented below each pretreatment. The right panel depicts subtypes of RNA classes accessible for 3′-end tailing (-oligo(A) or –oligo(C) tailing) and adapter ligation. (C) Possible biases associated with RNA 5′-(left) and 3′-(right) end-modifications and with the subsequent steps of cDNA construction.

Figure 2.

Schematic representation of the three-step mechanism of the bacteriophage T4 RNA ligase (T4Rnl) reaction with the potential side products. Encircled Arabic numbers indicate the order of ligation steps. Step 1: adenylation of the T4Rnl active site; Step 2: donor 5′-adenylation (side reaction: circularization of 3′-end-unprotected donor molecules); Step 3: phosphodiester bond formation between 3′-hydroxylated (OH) acceptor and donor molecules (side reaction: reverse adenylation of donor and circularization of acceptor molecules). PPi: pyrophosphate, p: monophosphate, AMP: adenosine monophosphate (Ap), App: 5′-adenylated termini.