RNA-Seq benchmarking study: A comparison of Element and Illumina sequencing platforms at the UMGC

December 12, 2024

The UMN Genomics Center (UMGC) has acquired a suite of AVITI sequencing systems by Element Biosciences to give researchers expanded options for short-read, mid-throughput sequencing. As with any new technology adopted at the UMGC, it is essential to assess their performance with specific library types, in this case, RNA-Seq libraries prepared from two commonly used kits at the UMGC. The aim of this study was to evaluate and compare quality metrics and gene expression counts for identical samples sequenced using the Element AVITI and Illumina NovaSeq X Plus platforms.

Materials and methods:
An identical set of 64 samples were sequenced on both sequencing platforms. Samples consisted of mouse tissue samples from three client-submitted UMGC projects, summarized here. cDNA reads were processed using the rnaseq-pipeline, which is part of gopher-pipelines module v2.5. This pipeline analyzes a set of RNA-Seq FASTQ files using HISAT2 v2.1.0 and Subread v1.6.3 featureCountsaligned. The Mus musculus reference genome GRCm39 was used for alignment.

Table RNA library prep methods — **Table 1:** Number of samples in each project.

Results:
AVITI base quality scores are slightly higher than Illumina. Mean and median Q scores were slightly higher on the AVITI platform than on the NovaSeq X Plus platform. The percentages of unique reads and the percentages of reads aligning to the reference genome were slightly higher with AVITI sequencing. The library prep methods used in this study produced 158-bp read lengths on reads the NovaSeq X Plus, whereas the AVITI produced 150-bp read lengths. This difference may explain the slight differences in the percentage of unique reads and the percentage of reads aligning to the reference genome.

**Figure 1.** Base quality scores averaged across all bases of all reads in a sample for Read 1 (A) and Read 2 (B) in Element AVITI vs. Illumina NovaSeq X Plus platforms. In these plots, each dot is one of the 64 samples in the library pool.

Gene expression counts between platforms are highly correlated. Comparisons of gene expression counts between the AVITI and NovaSeq X Plus datasets revealed strong correlations. The sample with the highest correlation, represented in the plot on the right below, showed an r-value of 0.975, while the sample with the lowest correlation, on the left, still exhibited a high r-value of 0.846.

**Figure 2.** Samples with the smallest (left) and largest (right) r-values. Reads per million (RPM)-transformed gene counts were log10 transformed after adding a pseudocount equal to half of the smallest RPM-transformed value. Pearson correlation tests were performed between these transformed gene abundances detected by both sequencing platforms.

When considering all 64 samples, the analysis demonstrated high correlation values across the dataset.

**Figure 3.** R-value analysis between paired samples sequenced using Element AVITI vs. Illumina NovaSeq X Plus platforms. Reads per million (RPM)-transformed gene counts were log10 transformed after adding a pseudocount equal to half of the smallest RPM-transformed value before Pearson correlation tests were performed.

Conclusions:
Paired-end 150-bp sequencing on the AVITI platform produced comparable results to paired-end 158-bp sequencing in the Illumina NovaSeq X Plus platform. Quality scores were slightly higher on the AVITI platform compared to the NovaSeq X Plus platform. This difference is marginal and unlikely to necessitate modifications to standard RNA-Seq analysis workflows. The UMGC does not recommend switching sequencing platforms midway through an ongoing RNA-Seq project. It is advisable to complete such projects using Illumina data before transitioning to the AVITIs for subsequent RNA-Seq experiments.

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