Dual randomization of oligonucleotides to reduce the bias in ribosome-profiling libraries

Abstract

Protein translation is at the heart of cellular metabolism and its in-depth characterization is key for many lines of research. Recently, ribosome profiling became the state-of-the-art method to quantitatively characterize translation dynamics at a transcriptome-wide level. However, the strategy of library generation affects its outcomes. Here, we present a modified ribosome-profiling protocol starting from yeast, human cells and vertebrate brain tissue. We use a DNA linker carrying four randomized positions at its 5' end and a reverse-transcription (RT) primer with three randomized positions to reduce artifacts during library preparation. The use of seven randomized nucleotides allows to efficiently detect library-generation artifacts. We find that the effect of polymerase chain reaction (PCR) artifacts is relatively small for global analyses when sufficient input material is used. However, when input material is limiting, our strategy improves the sensitivity of gene-specific analyses. Furthermore, randomized nucleotides alleviate the skewed frequency of specific sequences at the 3' end of ribosome-protected fragments (RPFs) likely resulting from ligase specificity. Finally, strategies that rely on dual ligation show a high degree of gene-coverage variation. Taken together, our approach helps to remedy two of the main problems associated with ribosome-profiling data. This will facilitate the analysis of translational dynamics and increase our understanding of the influence of RNA modifications on translation.

Keywords: Codon-translation speed; RNA modification; Ribosome profiling; Sequencing bias; Translation; Translational control.

Fig. 1

Comparison of library preparation methods. (A) RPF coverage of representative yeast ORFs made with a commercial dual ligation kit or according to a circularization based approach. Coverage of circularization-based libraries is given in orange, dual ligation libraries in light blue (colors match in A, B, C and E). The annotated ORF is indicated in dark blue. (B) Boxplot showing standard deviations of codon coverage of individual transcripts in three replicates of the ribosome-profiling libraries shown in A. Sequencing density was corrected for library size and the first 15 codons in each gene were omitted from analysis. Higher values and wider boxplots point to uneven coverage of transcripts. (C) Principal Component Analysis (PCA) comparing transcript read counts from both library types using three replicates each. Top right: Regression analysis of transcript read counts from both libraries, regression line in red. Spearman correlation: 0.8771. (D) Differential expression of two-linker ligation libraries relative to circularization libraries (n = 3) using DESeq2 with a log2 fold change threshold of 0.8 and an adjusted p-value of 0.05. Differentially expressed genes are indicated in red. (E) Codon-specific A-site ribosome occupancy relative to downstream sites (mean ± SD, n = 3). Symbol size indicates the relative frequency of codons in the A-site.

Fig. 2

Schematic overview of ribosome-profiling library preparation. (A) Schematic overview of ribosome-profiling library preparation from footprint sequences (blue), using fixed or randomized linkers in combination with a standard or randomized RT primer. Illumina sequencing of the resulting libraries includes randomized sequences allowing for downstream analysis and artifact removal. (B) Example of a dual randomized sequencing read showing three randomized positions (3 N) derived from the RT-primer (green), the footprint (blue) and the four randomized positions (4 N) of the linker (purple). This allows to distinguish amplification duplicates (top) with identical randomized positions from biological duplicates with different barcodes (bottom).

Fig. 3

Identification of duplicates in ribosome-profiling reads. (A) Compositions of mRNA and RPF libraries prepared with fixed linkers or two different randomization strategies. Unique reads (blue) are sequences only present once in the library regardless of randomized sequences. Amplification duplicates (red) are reads that cannot be identified as unique based on randomization. The remaining sequences appear multiple times, but can be distinguished by randomization of the DNA linker (green), the RT primer (yellow) or a unique combination of both (orange). (B) Composition of two replicates of RPF libraries (as in A) with fixed linkers or dual randomization using a high input (samples as in A) or a 2–6× lower input of the same monosomal RNA for ribosome profiling. Library pairs (high:low) were randomly downsampled to an equal number of reads to accurately reflect differences in library composition.

Fig. 4

Sequence bias in ribosome-profiling libraries. (A) Frequencies of triplets along RPF reads. (B) End frequency vs. total frequency of triplets in fixed-linker (left) and dual-randomized (right) libraries. The midline (red) represents a perfect match of frequencies. (C) Distance of data points in (B) to the line midline. Triplets are ordered based on differences in randomized-linker libraries. Similar shapes represent samples derived from the same biological replicate.